Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase

ABSTRACT

The invention relates to a polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group, particularly when said BCN group is present as: a residue of a lysine amino acid. The invention also relates to a method of producing a polypeptide comprising a BCN group, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide. The invention also relates to an amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), particularly and amino acid which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. In addition the invention relates to a PylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.

FIELD OF THE INVENTION

The invention relates to site-specific incorporation of bio-orthogonalgroups via the (expanded) genetic code. In particular the inventionrelates to incorporation of chemical groups into polypeptides viaaccelerated inverse electron demand Diels-Alder reactions betweengenetically incorporated amino acid groups such as dienophiles, andchemical groups such as tetrazines.

BACKGROUND TO THE INVENTION

The site-specific incorporation of bio-orthogonal groups via geneticcode expansion provides a powerful general strategy for sitespecifically labelling proteins with any probe. However, the slowreactivity of the bio-orthogonal functional groups that can begenetically encoded has limited this strategy's utility.

The rapid, site-specific labeling of proteins with diverse probesremains an outstanding challenge for chemical biologists; enzymemediated labeling approaches may be rapid, but use protein or peptidefusions that introduce perturbations into the protein under study andmay limit the sites that can be labeled, while many ‘bio-orthogonal’reactions for which a component can be genetically encoded are too slowto effect the quantitative and site specific labeling of proteins on atime-scale that is useful to study many biological processes.

There is a pressing need for general methods to site-specifically labelproteins, in diverse contexts, with user-defined probes.

Inverse electron demand Diels-Alder reactions between strained alkenesincluding norbornenes and trans-cyclooctenes, and tetrazines haveemerged as an important class of rapid bio-orthogonal reactions¹⁻⁴. Therates reported for some of these reactions are incredibly fast^(3,4).

Very recently, three approaches have been reported for specificallylabeling proteins using these reactions:

-   -   A lipoic acid ligase variant that accepts a trans-cyclooctene        substrate has been used to label proteins bearing a 13 amino        acid lipoic acid ligase tag in a two step procedure⁵.    -   A tetrazine has been introduced at a specific site in a protein        expressed in E. coli via genetic code expansion, and derivatized        with a strained trans-cyclooctene-diacetyl fluorescein⁶.    -   The incorporation of a strained alkene (a norbornene containing        amino acid) has been demonstrated via genetic code expansion and        site-specific fluorogenic labeling with tetrazine fluorophores        in vitro, in E. coli and on mammalian cells⁷. The incorporation        of norbornene containing amino acids has also been recently        reported.^(8,9)

The low-efficiency incorporation of a trans-cycclooctene containingamino acid (TCO) (2) has been reported, with detection of somefluorescent labelling in fixed cells.⁹

Recent work with model reactions in organic solvents suggests that thereaction between BCN (first described in strain promoted reactions withazides)¹⁰ and tetrazines may proceed very rapidly¹¹. However, thisreaction, unlike the much slower reaction of simple cyclooctynes withazides, nitrones¹²⁻¹⁶ and tetrazines^(9,17), has not been explored inaqueous media or as a chemoselective route to labeling macromolecules.

The present invention seeks to overcome problem(s) associated with theprior art.

SUMMARY OF THE INVENTION

Certain techniques for the attachment of tetrazine compounds topolypeptides exist in the art. However, those techniques suffer fromslow reaction rates. Moreover, those techniques allow for multiplechemical species to be produced as reaction products. This can lead toproblems, for example in variable molecular distances between dye groupswhich can be problematic for fluorescence resonance energy transfer(FRET) analysis. This can also be problematic for the production oftherapeutic molecules since heterogeneity of product can be a drawbackin this area.

The present inventors have provided a new amino acid bearing abicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group. This allows adramatically increased reaction rate, which is advantageous. Inaddition, this allows a single-product addition reaction to be carriedout. This leads to a homogeneous product, which is an advantage. Thisalso eliminates isomeric variations (spatial isomers) in the product,which provides technical benefits in a range of applications asdemonstrated herein. In addition, the product of the BCN additionreaction does not epimerise, whereas the products from (for example)norbornene and/or TCO reactions do give rise to epimers. Thus it is anadvantage of the invention that the problems of epimers are alsoavoided.

Thus in one aspect the invention provides a polypeptide comprising anamino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.This has the advantage of providing a single reaction product followingaddition of (for example) tetrazine compounds. Alternate techniques suchas norbornene addition or TCO addition give a mixture of productscomprising different isomers, such as regio or stereo isomers. Onereason for this advantage is that the BCN part of the molecule hasmirror symmetry so that the product is the same, whereas forTCO/norbornene that part of the molecule is chiral and so attachment canbe to the ‘top face’ or ‘bottom face’ of the double bond, leading todifferent isomers in the products.

Thus the invention provides the advantage of homogeneity of product whenused in the attachment of further groups to the polypeptide such astetrazine compounds.

Suitably said BCN group is present as a residue of a lysine amino acid.

In another aspect, the invention relates to a method of producing apolypeptide comprising a BCN group, said method comprising geneticallyincorporating an amino acid comprising a BCN group into a polypeptide.

Suitably producing the polypeptide comprises

(i) providing a nucleic acid encoding the polypeptide which nucleic acidcomprises an orthogonal codon encoding the amino acid having a BCNgroup;

(ii) translating said nucleic acid in the presence of an orthogonal tRNAsynthetase/tRNA pair capable of recognising said orthogonal codon andincorporating said amino acid having a BCN group into the polypeptidechain.

Suitably said amino acid comprising a BCN group is a BCN lysine.

Suitably said orthogonal codon comprises an amber codon (TAG), said tRNAcomprises MbtRNA_(CUA). Suitably said amino acid having a BCN groupcomprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. Suitablysaid tRNA synthetase comprises a PylRS synthetase having the mutationsY271M, L274G and C313A (BCNRS).

Suitably said amino acid having a BCN group is incorporated at aposition corresponding to a lysine residue in the wild type polypeptide.This has the advantage of maintaining the closest possible structuralrelationship of the BCN containing polypeptide to the wild typepolypeptide from which if is derived.

In another aspect, the invention relates to a polypeptide as describedabove which comprises a single BCN group. Thus suitably the polypeptidecomprises a single BCN group. This has the advantage of maintainingspecificity for any further chemical modifications which might bedirected at the BCN group. For example when there is only a single BCNgroup in the polypeptide of interest then possible issues of partialmodification (e.g. where only a subset of BCN groups in the polypeptideare subsequently modified), or issues of reaction microenvironmentsvarying between alternate BCN groups in the some polypeptides (whichcould lead to unequal reactivity between different BCN group(s) atdifferent locations in the polypeptide) are advantageously avoided.

A key advantage of incorporation of a BCN group is that is permits arange of extremely useful further compounds such as labels to be easilyand specifically attached to the BCN group.

In another aspect, the invention relates to a polypeptide as describedabove wherein said BCN group is joined to a tetrazine group.

In another aspect, the invention relates to a polypeptide as describedabove wherein said tetrazine group is further joined to a fluorophore.

Suitably said fluorophore comprises fluorescein, tetramethyl rhodamine(TAMRA) or boron-dipyrromethene (BODIPY).

In another aspect, the invention relates to a novel unnatural amino acidcomprising a BCN group.

In another aspect, the invention relates to an amino acid comprisingbicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN).

In another aspect, the invention relates to an amino acid which isbicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine.

Suitably BCN lysine as described above has the structure:

In another aspect, the invention relates to a method of producing apolypeptide comprising a tetrazine group, said method comprisingproviding a polypeptide as described above, contacting said polypeptidewith a tetrazine compound, and incubating to allow joining of thetetrazine to the BCN group by on inverse electron demand Diels-Aldercycloaddition reaction.

Suitably the tetrazine is selected from 6 to 17 of FIG. 1.

Suitably the pseudo first order rate constant for the reaction is atleast 80 M⁻¹ s⁻¹.

Suitably the tetrazine is selected from 6, 7, 8 and 9 of FIG. 1 and thepseudo first order rate constant for the reaction is at least 80 M⁻¹s⁻¹.

This chemistry has the advantage of speed of reaction.

Suitably said reaction is allowed to proceed for 10 minutes or less.

Suitably said reaction is allowed to proceed for 1 minute or less.

Suitably said reaction is allowed to proceed for 30 seconds or less.

It will be noted that certain reaction environments may affect reactiontimes. Most suitably the shortest times such as 30 seconds or less areapplied to in vitro reactions.

Reactions in vivo, or in eukaryotic culture conditions such as tissueculture medium or other suitable media for eukaryotic cells, may need tobe conducted for longer than 30 seconds to achieve maximal labelling.The skilled operator can determine optimum reaction times by trial anderror based on the guidance provided herein.

Suitably said tetrazine compound is a tetrazine compound selected fromthe group consisting of 11 and 17 of FIG. 1.

In another aspect, the invention relates to a PylRS tRNA synthetasecomprising the mutations Y271M, L274G and C313A.

Suitably said PylRS tRNA synthetase has a sequence corresponding toMbPylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.

In another aspect the invention relates to the use of the PylRS tRNAsynthetase(s) of the invention for the incorporation of amino acidcomprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) into a polypeptide.

In another aspect the invention relates to a method for theincorporation of amino acid comprisingbicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) into a polypeptide comprisinguse of the PylRS tRNA synthetase(s) of the invention to incorporatesame.

In another aspect, the invention relates to a homogenous recombinantpolypeptide as described above. Suitably said polypeptide is made by amethod as described above.

Also disclosed is a polypeptide produced according to the method(s)described herein. As well as being the product of those new methods,such a polypeptide has the technical feature of comprising BCN.

Mutating has it normal meaning in the art and may refer to thesubstitution or truncation or deletion of the residue, motif or domainreferred to. Mutation may be effected at the polypeptide level e.g. bysynthesis of a polypeptide having the mutated sequence, or may beeffected at the nucleotide level e.g. by making a nucleic acid encodingthe mutated sequence, which nucleic acid may be subsequently translatedto produce the mutated polypeptide. Where no amino acid is specified asthe replacement amino acid for a given mutation site, suitably arandomisation of said site is used. As a default mutation, alanine (A)may be used. Suitably the mutations used at particular site(s) are asset out herein.

A fragment is suitably at least 10 amino acids in length, suitably atleast 25 amino acids, suitably at least 50 amino acids, suitably atleast 100 amino acids, suitably at least 200 amino acids, suitably atleast 250 amino acids, suitably at least 300 amino acids, suitably atleast 313 amino acids, or suitably the majority of the polypeptide ofinterest.

DETAILED DESCRIPTION OF THE INVENTION

Here we demonstrate a fluorogenic reaction betweenbicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines. The rates forthese reactions are 3-7 orders of magnitude faster than the rates formany ‘bio-orthogonal’ reactions. We describe aminoacyl-tRNAsynthetase/tRNA pairs and their use for the efficient site-specificincorporation of a BCN-containing amino acid, 1, and atranscyclooctene-containing amino acid 2 (which also reacts extremelyrapidly with tetrazines) into proteins expressed in E. coli andmammalian cells. We demonstrate the site-specific, fluorogenic labelingof proteins containing 1 and 2 in vitro, in E. coli and in livemammalian cells at the first measurable time point (after seconds orminutes). Moreover we demonstrate the specificity of tetrazine labelingwith respect to a proteome as well as the advantages of the approachwith respect to current ‘bio-orthogonal’ reactions for which a componentcan be encoded. The approaches developed may be applied to site-specificprotein labeling in animals, and they find utility in labelling andimaging studies.

A polypeptide comprising an amino acid having a dienophile group,characterised in that said dienophile group comprises abicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.

We describe genetic encoding of bicyclononynes and trans-cyclooctenesfor site-specific protein labelling in vitro and in live mammalian cellsvia fluorogenic Diels-Alder reactions.

The methods of the invention may be practiced in vivo or in vitro.

In one embodiment, suitably the methods of the invention are not appliedto the human or animal body. Suitably the methods of the invention arein vitro methods. Suitably the methods do not require the presence ofthe human or animal body. Suitably the methods are not methods ofdiagnosis or of surgery or of therapy of the human or animal body.

Dienophile/Trans-Cyclooctene (TCO) Aspects

In a broad aspect the invention relates to a polypeptide comprising anamino acid having a dienophile group capable of reacting with atetrazine group.

Suitably said dienophile group is present as a residue of a lysine aminoacid.

In one embodiment, the invention relates to a method of producing apolypeptide comprising a dienophile group, said method comprisinggenetically incorporating an amino acid comprising a dienophile groupinto a polypeptide.

Suitably producing the polypeptide comprises

(i) providing a nucleic acid encoding the polypeptide which nucleic acidcomprises an orthogonal codon encoding the amino acid having adienophile group;

(ii) translating said nucleic acid in the presence of an orthogonal tRNAsynthetase/tRNA pair capable of recognising said orthogonal codon andincorporating said amino acid having a dienophile group into thepolypeptide chain. Suitably said amino acid comprising a dienophilegroup is a dienophile lysine.

Suitably said orthogonal codon comprises an amber codon (TAG), said tRNAcomprises MbtRNA_(CUA), said amino acid having a dienophile groupcomprises a trans-cyclooctene-4-ol (TCO) containing amino acid and saidtRNA synthetase comprises a PylRS synthetase having the mutations Y271A,L274M and C313A (TCORS).

Suitably said PylRS tRNA synthetase has a sequence corresponding toMbPylRS tRNA synthetase comprising the mutations Y271A, L274M and C313A(TCORS). In another aspect the invention relates to the use of the PylRStRNA synthetase(s) of the invention for the incorporation of amino acidcomprising trans-cyclooctene-4-ol (TCO) into a polypeptide.

In another aspect the invention relates to a method for theincorporation of amino acid comprising trans-cyclooctene-4-ol (TCO) intoa polypeptide comprising use of the PylRS tRNA synthetase(s) of theinvention to incorporate same.

Aspects of the invention regarding the joining of tetrazine compounds tothe unnatural amino acids discussed herein apply equally to TCO aminoacids as they do to BCN amino acids unless otherwise indicated by thecontext.

We report the exceptionally rapid, fluorogenic, reaction of BCN with arange of tetrazines under aqueous conditions at room temperature. Therate constants for BCN-tetrazine reactions are 500 to 1000 times greaterthan for the reaction of norbornene with the same tetrazines. The rateconstants for TCO-tetrazine reactions are 10-15 fold greater than thosefor BCN with the same tetrazine. The reaction between strained alkenesand tetrazines may lead to a mixture of diastereomers and regioisomers,as well as isomers from dihydropyridazine isomerization.^(3,4)

In contrast the BCN tetrazine reaction leads to the formation of asingle product. This may be an advantage in applications wherehomogeneity in the orientation of probe attachment may be important,including single molecule spectroscopy, and FRET approaches.

We have described aminoacyl-tRNA synthetase/tRNA pairs and their uses todirect the efficient, site-specific incorporation of 1 and 2 intoproteins in E. coli and mammalian cells.

We have demonstrated that the specific, quantitative labeling ofproteins—a process that takes tens of minutes to hours with an encodednorbornene⁷ and tens of hours with an encoded azide usingcopper-catalysed click chemistry with alkyne probes²¹—may be completewithin seconds using the encoded amino acids 1 and 2. While we do notobserve labeling of an azide incorporated into EGFR on the mammaliancell surface with cyclooctynes⁷ and labeling of an encoded norbornene inEGFR allows labeling only after 2 hours with tetrazines⁷, strong andsaturated labeling of EGFR incorporating 1 and 2 was observed at thefirst time point measured (2 min) using nanomolar concentrations oftetrazine-dye conjugates. These experiments confirm that the rapidBCN-tetrazine and TCO-tetrazine ligations characterized in smallmolecule experiments translate into substantial improvements in proteinlabeling in diverse contexts. While we have demonstrated the advantagesof this approach in vitro, in E. coli and in live mammalian cells theability to incorporate unnatural amino acids in C. elegans using thePylRS/tRNA_(CUA) pair²⁹ suggests that it may be possible to extend thelabeling approach described here to site-specific protein labeling inanimals.

Genetic Incorporation and Polypeptide Production

In the method according to the Invention, said genetic incorporationpreferably uses an orthogonal or expanded genetic code, in which one ormore specific orthogonal codons have been allocated to encode thespecific amino acid residue with the BCN group so that it can begenetically incorporated by using on orthogonal tRNA synthetase/tRNApair. The orthogonal tRNA synthetase/tRNA pair can in principle be anysuch pair capable of charging the tRNA with the amino acid comprisingthe BCN group and capable of incorporating that amino acid comprisingthe BCN group into the polypeptide chain in response to the orthogonalcodon.

The orthogonal codon may be the orthogonal codon amber, ochre, opal or aquadruplet codon. The codon simply has to correspond to the orthogonaltRNA which will be used to carry the amino acid comprising the BCNgroup. Preferably the orthogonal codon is amber.

It should be noted that the specific examples shown herein have used theamber codon and the corresponding tRNA/tRNA synthetase. As noted above,these may be varied. Alternatively, in order to use other codons withoutgoing to the trouble of using or selecting alternative tRNA/tRNAsynthetase pairs capable of working with the amino acid comprising theBCN group, the anticodon region of the tRNA may simply be swapped forthe desired anticodon region for the codon of choice. The anticodonregion is not involved in the charging or incorporation functions of thetRNA nor recognition by the tRNA synthetase so such swaps are entirelywithin the ambit of the skilled operator.

Thus alternative orthogonal tRNA synthetase/tRNA pairs may be used ifdesired.

Preferably the orthogonal synthetase/tRNA pair are Methanosarcinabarkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate ambersuppressor tRNA (MbtRNA_(CUA)).

The Methanosarcina barkeri PylT gene encodes the MbtRNA_(CUA) tRNA.

The Methanosarcina barkeri PylS gene encodes the MbPylRS tRNA synthetaseprotein. When particular amino acid residues are referred to usingnumeric addresses, the numbering is taken using MbPylRS (Methanosarcinabarkeri pyrrolysyl˜tRNA synthetase) amino acid sequence as the referencesequence (i.e. as encoded by the publicly available wild typeMethanosarcina barkeri PylS gene Accession number Q46E77):

MDKKPLDVLI SATGLWMSRT GTLHKIKHYE VSRSKIYIEMACGDHLVVNN SRSCRTARAF RHHKYRKTCK RCRVSDEDINNFLTRSTEGK TSVKVKVVSA PKVKKAMPKS VSRAPKPLENPVSAKASTDT SRSVPSPAKS TPNSPVPTSA PAPSLTRSQLDRVEALLSPE DKISLNIAKP FRELESELVT RRKNDFQRLYTNDREDYLGK LERDITKFFV DRDFLEIKSP ILIPAEYVERMGINNDTELS KQIFRVDKNL CLRPMLAPTL YNYLRKLDRILPDPIKIFEV GPCYRKESPG KEHLEEFTMV NFCQMGSGCTRENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDIMHGDLELSSAVVGPV PLDREWGIDK PWIGAGFGLE RLLKVMHGFK NIKRASRSES YYNGISTNL.

Said sequence has been annotated here below as SEQ ID NO. 1.

If required, the person skilled in the art may adapt MbPylRS tRNAsynthetase protein by mutating it so as to optimise for the BCN aminoacid to be used. The need for mutation depends on the BCN amino acidused. An example where the MbPylRS tRNA synthetase may need to bemutated is when the BCN amino acid is not processed by the MbPylRS tRNAsynthetase protein.

Such mutation may be carried out by introducing mutations into theMbPylRS tRNA synthetase, for example at one or more of the followingpositions in the MbPylRS tRNA synthetase: M241, A267, Y271, L274 andC313.

An example is when said amino acid having a BCN group comprises abicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. Suitably said tRNAsynthetase comprises a PylRS synthetase such as MbPylRS having themutations Y271M, L274G and C313A (BCNRS).

An example is when said amino acid having a dienophile group comprises atrans-cyclooctene-4-ol (TCO) containing amino acid. Suitably said tRNAsynthetase comprises a PylRS synthetase such as MbPylRS having themutations Y271A, L274M and C313A (TCORS).

tRNA Synthetases

The tRNA synthetase of the invention may be varied. Although specifictRNA synthetase sequences may have been used in the examples, theinvention is not intended to be confined only to those examples.

In principle any tRNA synthetase which provides the same tRNA charging(aminoacylation) function can be employed in the invention.

For example the tRNA synthetase may be from any suitable species such asfrom archea, for example from Methanosarcina barkeri MS; Methanosarcinabarkeri sir, Fusaro; Methanosarcina mazei Gol; Methanosarcinaacetivorans C2A; Methanosarcina thermophila; or Methanococcoidesburtonii. Alternatively the tRNA synthetase may be from bacteria, forexample from Desulfitobacterium hafniense DCB-2; Desulfitobacteriumhafniense Y51; Desulfitobacterium hafniense PCP1; Desulfotomaculumacetoxidans DSM 771.

Exemplary sequences from these organisms are the publically availablesequences. The following examples are provided as exemplary sequencesfor pyrrolysine tRNA synthetases:

>M. barkeriMS/1-419/ Methanosarcina barkeri MSVERSION Q6WRH6.1 GI: 74501411MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL>M. barkeriF/1-419/ Methanosarcina barkeri str. FusaroVERSION YP_304395.1 GI: 73668380MDKKPLDVLISATGLWMSRTGTLHKIKHYEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVSAPKVKKAMPKSVSRAPKPLENPVSAKASTDSRSVPSPAKSTPNSPVPTSAPAPSLTRSQLDRVEALLSPEDKISLNIAKPFRELESELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRDFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPDPIKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLESLIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVPLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL>M. mazei/1-454 Methanosarcina mazei Go1VERSION NP_63346931 GI: 21227547MDKKPLNTLISATGIWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSSTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL >M. acetivorans/1-443Methanosarcina acetivorans C2A VERSION NP_615128.2 GI: 161484944MDKKPLDTLISATGLWMSRTGMIHKIKHHEVSRSKIYIEMACGERLVVNNSRSSRTARALRHHKYRKTCRHCRVSDEDINNFLTKTSEEKTTVKVVSAPRVRKAMPKSVARAPKPLEATAQVPLSGSKPAPATPVSAPAQAPAPSTGSASATSASAQRMANSAAAPAAPVPTSAPALTKGQLDRLEGLLSPKDEISLDSEKPFRELESELLSRRKKDLKRIYAEERENYLGKLEREITKFFVDRGFLEIKSPILIPAEYVERMGINSDTELSKQVFRIDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLEAIITEFLNHLGIDFEIIGDSCMVYGNTLDVMHDDLELSSAVVGPVPLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRAARSESYYNGISTNL >M. thermophila/1-478Methanosarcina thermophila VERSION DQ017250.1 GI: 67773308MDKKPLNTUSATGLWMSRTGKLHKIRHHEVSKRKIYIEMECGERLVVNNSRSCRAARALRHHKYRKICKHCRVSDEDLNKFLTRTNEDKSNAKVTVVSAPKIRKVMPKSVARTPKPLENTAPVQTLPSESQPAPTTPISASTTAPASTSTTAPAPASTTAPAPASTTAPASASTTISTSAMPASTSAQGTTKFNYISGGFPRPIPVQASAPALTKSQIDRLQGLLSPKDEISLDSGTPFRKLESELLSRRRKDLKQIYAEEREHYLGKLEREITKFFVDRGFLEIKSPILIPMEYIERMGIDNDKELSKQIFRVDNNFCLRPMLAPNLYNYLRKLNRALPDPIKTFBGPCYRKESDGKEHLEERMLNFCQMGSGCTRENEAHKDFLDYLGIDFEIVGDSCMVYGDTLDVMHGDLELSSAVVGPVPMDRDWGINKPWIGAGFGLERLLKVMHNFICNIKRASRSESYYNGISTNL >M. burtonii/1-416Methanococcoides burtonii DSM 6242 VERSION YP_566710.1 GI: 91774018MEKQLLDVLVELNGVWLSRSGLLHGIRNFEINKHIHIETDCGARFTVRNSRSSRSARSLRHNKYRKPCKRCRPADEQIDRFVKKTFKEKRQTVSVFSSPKKHVPKKPKVAVKSFSISTPSPKEASVSNSIPTPSISVVKDEVKVPEVKYTPSQIERLKTLMSPDDKIPIQDELPEFKVLEKELFQRRRDDLKKMYEEDREDRLGKLERDITEFFVDRGFLEIKSPIMIPFEYIERMGIDKDDHLNKQIFRVDESMCLRPMLAPCLYNYLRKLDKVLPDPIRIFEIGPCYRKESDGSSHLEEFTMVNFCQMGSGCTRENMEALIDEFLEHLGIEYEIEADNCMVYGDTIDIMHGDLELSSAVVGPIPLDREWGVNICPVVMGAGFGLERUKVRHNYTNIRRASRSELYYNGINTNL>D. hafniense_DCB-2/1-279 Desulfitobacterium hafniense DCB-2VERSION YP_002461289.1 GI: 219670854MSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLELEEGLAKALHQQGFVQVVTPTIITKSALAKMTIGEDHPLFSQVFWLDGKKCLRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTMLNTELGTPLEERHQRLEDMARWVLEAAGIREFELVTESSVVYGDTVDVMKGDLELASGAMGPHFLDEKWEIVDPWVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN >D. hafniense_Y51/1-312 Desulfitobacterium hanfniense Y51VERSION YP_521192.1 GI: 89897705MDRIDHTDSKFVQAGETPVLPATFMFLTRRDPPLSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLELEEGLAKALHQQGFVQVVTPTIITSKALAKMTIGEDHPLFSQVFWLDGKKCLRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTMLNTELGTPLEERHQRLEDMARWVLEAAGIREFELVTESSVVYGDTVDVMKGDLELASGAMGPHFLDEKWEIVDPWVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN >D. hafniensePCP1/1-288Desulfitobacterium hafniense VERSION AY692340.1 GI: 53771772MFLTRRDPPLSSFWTKVQYQRLKELNASGEQLEMGFSDALSRDRAFQGIEHQLMSQGKRHLEQLRTVKHRPALLELEEKLAKALHQQGFVQVVTPTIITKSALAKMTIGEDHPLFSQVFWLDGKKCLRPMLAPNLYTLWRELERLWDKPIRIFEIGTCYRKESQGAQHLNEFTMLNLTELGTPLEERHQRLEDMARWVLEAAGIREFELVTESSVVYGDTVDVMKGDLELASGAMGPHFLDEKWEIFDPWVGLGFGLERLLMIREGTQHVQSMARSLSYLDGVRLNIN >D. acetoxidans/1-277Desulfotomaculum acetoxidans DSM 771VERSION YP_003189614.1 GI: 258513392MSFLWTVSQQKRLSELNASEEEKNMSFSSTSDREAAYKRVEMRLINESKQRLNKLRHETRPAICALENRLAAALRGAGFVQVATPVILSKKLLGKMTITDEHALFSQVFWIEENKCLRPMLAPNLYYILKDLLRLWEKPVRIFEIGSCFRKESQGSNHLNEFTMLNLVEWGLPEEQRQKRISELAKLVMDETGIDEYHLEHAESVVYGETVDVMHRDIELGSGALGPHFLDGRWGVVGPWVGIGFGLERLLMVEQGGQNVRSMGKSLTYLDG VRLNI

When the particular tRNA charging (aminoacylation) function has beenprovided by mutating the tRNA synthetase, then it may not be appropriateto simply use another wild-type tRNA sequence, for example one selectedfrom the above. In this scenario, it will be important to preserve thesame tRNA charging (aminoacylation) function. This is accomplished bytransferring the mutation(s) in the exemplary tRNA synthetase into analternate tRNA synthetase backbone, such as one selected from the above.

In this way it should be possible to transfer selected mutations tocorresponding tRNA synthetase sequences such as corresponding pylSsequences from other organisms beyond exemplary M. barkeri and/or M.mazei sequences.

Target tRNA synthetase proteins/backbones, may be selected by alignmentto known tRNA synthetases such as exemplary M. barkeri and/or M. mazeisequences.

This subject is now illustrated by reference to the pylS (pyrrolysinetRNA synthetase) sequences but the principles apply equally to theparticular tRNA synthetase of interest.

For example, FIG. 6 provides an alignment of all PylS sequences. Thesecan have a low overall % sequence identity. Thus it is important tostudy the sequence such as by aligning the sequence to known tRNAsynthetases (rather than simply to use a low sequence identity score) toensure that the sequence being used is indeed a tRNA synthetase.

Thus suitably when sequence identity is being considered, suitably it isconsidered across the tRNA synthetases as in FIG. 6. Suitably the %identity may be as defined from FIG. 6. FIG. 7 shows a diagram ofsequence identities between the tRNA synthetases. Suitably the %identity may be as defined from FIG. 7.

It may be useful to focus on the catalytic region. FIG. 8 aligns justthe catalytic regions. The aim of this is to provide a tRNA catalyticregion from which a high % identity can be defined to capture/identifybackbone scaffolds suitable for accepting mutations transplanted inorder to produce the same tRNA charging (aminoacylation) function, forexample new or unnatural amino acid recognition.

Thus suitably when sequence identity is being considered, suitably it isconsidered across the catalytic region as in FIG. 8. Suitably the %identity may be as defined from FIG. 8. FIG. 9 shows a diagram ofsequence identities between the catalytic regions. Suitably the %identity may be as defined from FIG. 9.

‘Transferring’ or ‘transplanting’ mutations onto an alternate tRNAsynthetase backbone can be accomplished by site directed mutagenesis ofa nucleotide sequence encoding the tRNA synthetase backbone. Thistechnique is well known in the art. Essentially the backbone pylSsequence is selected (for example using the active site alignmentdiscussed above) and the selected mutations are transferred to (i.e.made in) the corresponding/homologous positions.

When particular amino acid residues are referred to using numericaddresses, unless otherwise apparent, the numbering is taken usingMbPylRS (Methanosarcina barkeri pyrrolysyl-tRNA synthetase) amino acidsequence as the reference sequence (i.e. as encoded by the publiclyavailable wild type Methanosarcina barkeri PylS gene Accession numberQ46E77):

MDKKPLDVLI SATGLWMSRT GTLHKIKHYE VSRSKIYIEMACGDHLVVNN SRSCRTARAF RHHKYRKTCK RCRVSDEDINNFLTRSTEGK TSVKVKVVSA PKVKKAMPKS VSRAPKPLENPVSAKASTDT SRSVPSPAKS TPNSPVPTSA PAPSLTRSQLDRVEALLSPE DKISLNIAKP FRELESELVT RRKNDFQRLYTNDREDYLGK LERDITKFFV DRDFLEIKSP ILIPAEYVERMGINNDTELS KQIFRVDKNL CLRPMLAPTL YNYLRKLDRILPDPIKIFEV GPCYRKESDG KEHLEEFTMV NFCQMGSGCTRENLESLIKE FLDYLEIDFE IVGDSCMVYG DTLDIMHGDLELSSAVVGPV PLDREWGIDK PWIGAGFGLE RLLKVMHGFK NIKRASRSES YYNGISTNL

This is to be used as is well understood in the art to locate theresidue of interest. This is not always a strict countingexercise—attention must be paid to the context or alignment. Forexample, if the protein of interest is of a slightly different length,then location of the correct residue in that sequence corresponding to(for example) L266 may require the sequences to be aligned and theequivalent or corresponding residue picked, rather than simply takingthe 266th residue of the sequence of interest. This is well within theambit of the skilled reader.

Notation for mutations used herein is the standard in the art. Forexample L266M means that the amino acid corresponding to L at position266 of the wild type sequence is replaced with M.

The transplantation of mutations between alternate tRNA backbones is nowillustrated with reference to exemplary M. barkeri and M. mazeisequences, but the same principles apply equally to transplantation ontoor from other backbones.

For example Mb AcKRS is an engineered synthetase for the incorporationof AcK Parental protein/backbone: M. barkeri PylS

Mutations: L266V, L270I, Y271F, L274A, C317F

Mb PCKRS: engineered synthetase for the Incorporation of PCK

Parental protein/backbone: M. barkeri PylS

Mutations: M241F, A267S, Y271C, L274M

Synthetases with the same substrate specificities can be obtained bytransplanting these mutations into M. mazei PylS. The sequence homologyof the two synthetases con be seen in FIG. 10. Thus the followingsynthetases may be generated by transplantation of the mutations fromthe Mb backbone onto the Mm tRNA backbone: Mm AcKRS introducingmutations L301V, L305I, Y306F, L309A, C348F into M. mazei PylS,

and

Mm PCKRS introducing mutations M276F, A302S, Y306C, L309M into M. mazeiPylS.

Full length sequences of these exemplary transplanted mutationsynthetases are given below.

>Mb_PyIS/1-419MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL >Mb_AcKRS/1-419MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSGEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKCASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPIUPAEYVERMGINNDTELSKQIFRVDKNLCLRPMVAPTIFNYARKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFFQMGSGCTRENLEAUKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL >Mb_PCKRS/1-419MDKKPLDVLISSATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNOREDYLGKLERDITKFFVDRGFLEIKSPIUPAEYVERFGINNDTELSKQIFRVDKNLCLRPMLSPTLCNYMRKLORILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGOLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL >Mm_PyIS/1-454MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSWYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSGTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFGQMGSGCTRENLESIITDFLNHLGDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL >Mm_AcKRS/1-454MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEOQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYEIRMGIDNDTELSKQIFRVDKNFCLRPMVAPNIFNYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL >Mm_PCKRS/1-454MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTICSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERFGIDNDTELSKQIFRVDKNFCLRPMLSPNLCNYMRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAWGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL

The same principle applies equally to other mutations and/or to otherbackbones.

Transplanted polypeptides produced in this manner should advantageouslybe tested to ensure that the desired function/substrate specificitieshave been preserved.

Polynucleotides encoding the polypeptide of interest for the methoddescribed above can be incorporated into a recombinant replicablevector. The vector may be used to replicate the nucleic acid in acompatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells includebacteria such as E. coli.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence that is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

Vectors of the invention may be transformed or transfected into asuitable host cell as described to provide for expression of a proteinof the invention. This process may comprise culturing a host celltransformed with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid. Vectorsmay be used, for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding the protein ofthe invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell for which the expression vector isdesigned to be used in. The term promoter is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

Another aspect of the invention is a method, such as an in vitro method,of incorporating the BCN containing amino acid(s) genetically andsite-specifically into the protein of choice, suitably in a eukaryoticcell. One advantage of incorporating genetically by said method is thatit obviates the need to deliver the proteins comprising the BCN aminoacid into a cell once formed, since in this embodiment they may besynthesised directly in the target cell. The method comprises thefollowing steps:

-   i) introducing, or replacing a specific codon with, an orthogonal    codon such as an amber codon at the desired site in the nucleotide    sequence encoding the protein-   ii) introducing an expression system of orthogonal tRNA    synthetase/tRNA pair in the cell, such as a pyrollysyl-tRNA    synthetase/tRNA pair-   iii) growing the cells in a medium with the BCN containing amino    acid according to the invention.

Step (i) entails or replacing a specific codon with an orthogonal codonsuch as an amber codon at the desired site in the genetic sequence ofthe protein. This can be achieved by simply introducing a construct,such as a plasmid, with the nucleotide sequence encoding the protein,wherein the site where the BCN containing amino acid is desired to beintroduced/replaced is altered to comprise an orthogonal codon such asan amber codon. This is well within the person skilled in the art'sability and examples of such are given here below.

Step (ii) requires an orthogonal expression system to specificallyincorporate the BCN containing amino acid at the desired location (e.g.the amber codon). Thus a specific orthogonal tRNA synthetase such as anorthogonal pyrollysyl-tRNA synthetase and a specific correspondingorthogonal tRNA pair which are together capable of charging said tRNAwith the BCN containing amino acid are required. Examples of these areprovided herein.

Protein Expression and Purification

Host cells comprising polynucleotides of the invention may be used toexpress proteins of the invention. Host cells may be cultured undersuitable conditions which allow expression of the proteins of theinvention. Expression of the proteins of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, protein production can be initiated when required by, forexample, addition of an inducer substance to the culture medium, forexample dexamethasone or IPTG.

Proteins of the invention can be extracted from host cells by a varietyof techniques known in the art, including enzymatic, chemical and/orosmotic lysis and physical disruption.

Proteins of the invention can be purified by standard techniques knownin the art such as preparative chromatography, affinity purification orany other suitable technique.

DEFINITIONS

The term ‘comprises’ (comprise, comprising) should be understood to haveits normal meaning in the art, i.e. that the stated feature or group offeatures is included, but that the term does not exclude any otherstated feature or group of features from also being present.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structural formulae of unnatural amino acids 1 to 5 andtetrazine derivatives (6-17) used in this study. TAMRA-X, Bodipy TMR-X,Bodipy-FL and CFDA are common names for fluorophores; their structuralformulae are shown in Supplementary Figure S4).

FIG. 2 shows kinetic and spectrometric characterization of theBCN-tetrazine reaction. a) Stopped flow kinetics of the reaction; theinset shows the conjugation of tetrazine 7 to 5-norbornen-2-ol (Nor),note different timescales; conditions: c₇=0.05 mM, c_(BCN)=c_(Nor)=5 mMin MeOH/H_(2O) (55/45), 25° C. b) The second order rate constant k forthe reaction of 7 and BCN. c) The fluorogenic reaction of 11 with BCN.

FIG. 3 shows efficient, genetically encoded incorporation of unnaturalamino acids using the BCNRS/tRNA_(CUA) or TCORS/tRNA_(CUA) pair in E.coli. a) Amino acid dependent overexpression of sfGFP-His₆ bearing anamber codon at position 150. The expressed protein was detected inlysates using an anti-Hiss antibody. b) Coomassie stained gel showingpurified proteins. c-e) Mass spectrometry of amino acid incorporation:sfGFP-1-His₆. found: 28017.54 Da. calculated: 28017.62 Da; sfGFP-2-His₆.found: 27993.36 Da. calculated: 27992.82 Da; sfGFP-Hiss produced in thepresence of 3, as described in the text. found: 28019.34 Da. calculated:28019.63 Da. Smaller grey peaks in all mass spectra denote a loss of 131Da, which corresponds to the proteolytic cleavage of the N-terminalMethionine.

FIG. 4 shows rapid and specific labeling of recombinant proteins withtetrazine-fluorophores. a) Specific labeling of sfGFP bearing 1, 2 and 4with tetrazine-dye conjugate 11 (10 eq) demonstrated by SDS-PAGE andin-gel fluorescence. For sfGFP-His₆ produced in the presence of 3 onlyvery faint, sub-stoichiometric labeling is visible. b) Quantitativelabeling of sfGFP-1 with 11 demonstrated by ESI-MS (beforebioconjugation (blue spectrum. found: 28018.1±2 Da. calculated: 28017.6Da) and after bioconjugation (red spectrum. found 28824.2±2 Da.calculated: 28823.2 Da)). c) Quantitative labeling of sfGFP-2 with 11demonstrated by ESI-MS (before bioconjugation (blue spectrum. found:27993.2±2 Da. calculated: 27992.8 Da) and after bioconjugation (redspectrum. found 28799.4±2 Da. calculated: 28799.1 Da)). d) No labelingof sfGFP-His₆ (expressed in the presence of 3) with 11 could be detectedby MS. e) Very rapid labeling of proteins containing site-specificallyincorporated amino acid 1 and 2. sfGFP-1 (left) and sfGFP-2 (middle) arequantitatively labeled with 11 in the few seconds it takes to load thegel while it takes 1 h to completely label sfGFP-4 under the sameconditions (right).

FIG. 5 shows site specific incorporation of 1 and 2 into proteins inmammalian cells and the rapid and specific labeling of cell surface andintracellular mammalian proteins with 11. a) Western blots demonstratethat the expression of full length mCherry(TAG)eGFP-HA is dependent onthe presence of 1 or 2 and tRNA_(CUA). BCNRS, TCORS are FLAG tagged. b)Specific and ultra-rapid labeling of a cell surface protein in livemammalian cells. EGFR-GFP bearing 1, 2 or 5 at position 128 is visibleas green fluorescence at the membrane of transfected cells (leftpanels). Treatment of cells with 11 (400 nM) leads to selective labelingof EGFR that contains 1 or 2 (middle panels). Right panels show mergedgreen and red fluorescence images, DIC=differential interferencecontrast. Cells were imaged 2 minutes after the addition of 11. c)Specific and rapid labeling of a nuclear protein in live mammaliancells. Jun-1-mCherry is visible as red fluorescence in the nuclei oftransfected cells (left panels). Treatment of cells with the cellpermeable tetrazine dye 17 (200 nM) leads to selective labeling ofjun-1-mCherry (middle panel). Right panels show merged red and greenfluorescence. No labeling was observed for cells bearing jun-5-mCherry.

FIG. 6 shows alignment of PylS sequences.

FIG. 7 shows sequence identity of PylS sequences.

FIG. 8 shows alignment of the catalytic domain of PylS sequences (from350 to 480; numbering from alignment of FIG. 6).

FIG. 9 shows sequence identity of the catalytic domains of PylSsequences.

FIG. 10 shows alignment of synthetases with transplanted mutations basedon M. barkeri PylS or M. mazei PylS. The red asterisks indicate themutated positions.

FIG. 11 shows scheme 1. We demonstrate the synthesis, genetic encodingand fluorogenic labeling of unnatural amino acids 1 and 2 in vitro, inE. coli and in mammalian cells.

FIG. 12 (Supplementary Figure S1) shows LC/MS traces (254 nm) showingthe formation of pyridazine products (6-BCN, 7-BCN, 9-BCN, 8-BCN) fromreaction of the corresponding tetrazines (6, 7, 9 and 8) with 2equivalents of BCN (exo/endo mixture ˜4/1) in MeOH. All masses are givenin Daltons. The HPLC traces were taken after incubating the reactionsfor 10 to 30 minutes at room temperature. The overall yield forconversion to pyridazine products was >98%.

FIG. 13 (Supplementary Figure S2) shows determination of rate constantsk for the reaction of various tetrazines with BCN by UV-spectroscopyusing a stopped-flow device. (a) Response of the UV absorbance at 320 nmof compound 6 upon BCN addition (100 eq=5 mM); by fitting the data to asingle exponential equation, k′ values were determined (left panel);each measurement was carried out three to five times and the mean of theobserved rates k′ was plotted against the concentration of BCN to obtainthe rate constant k from the slope of the plot. For all four tetrazinescomplete measurement sets were done in duplicate (middle and rightpanel) and the mean of values is reported in Supplementary Table 1.(b-d) same as (a) for tetrazines 7, 9 and 8. Conditions:c_(tetrazine)=0.05 mM in 9/1 H₂O/MeOH, c_(BCN)=0.5 to 5 mM in MeOH,resulting in a final 55/45 MeOH/H₂O mixture. All experiments wererecorded at 25° C.

FIG. 14 (Supplementary Figure S3) shows determination of rate constantsk for the reaction of tetrazines 6 and 7 with TCO by UV-spectroscopyusing a stopped-flow device. (a) Response of the UV absorbance at 320 nmof compound 6 upon TCO addition (100 eq=5 mM): by fitting the data tothe sum of two single exponential equations, k′ values for the fastsingle exponential equations were determined (left panel): eachmeasurement was carried out three to five times and observed rates k′were plotted against the concentration of TCO to obtain the rateconstant k from the slope of the plot. For both tetrazines completemeasurement sets were done at least in duplicate (middle and rightpanel) and the mean of values is reported in Supplementary Table 1. (b)same as (a) for tetrazine 7. Conditions: c_(tetrazine)=0.05 mM in 9/1H₂O/MeOH, c_(TCO)=0.5 to 5 mM in MeOH, resulting in a final 55/45MeOH/H₂O mixture. All experiments were recorded at 25° C.

FIG. 15 (Supplementary Figure S4) shows structural formulae of varioustetrazine-fluorophores used in this study. Details on synthesis andcharacterization of these tetrazine-fluorophores can be found inreference 2.

FIG. 16 (Supplementary Figure S5) shows “Turn on” fluorescence oftetrazine—fluorophores upon reaction with9-hydroxymethylbicyclo[6.1.0]nonyne (BCN). A 2 microM solution of thecorresponding tetrazine-fluorophore in water (2 mM in DMSO) was reactedwith 300 equivalents of BCN. Emission spectra were recorded before and30 min after the addition of BCN. Excitation wavelengths: TAMRA-dyes andBodipy-TMR-X: 550 nm; Bodipy-FL: 490 nm.

FIG. 17 (Supplementary Figure S6) shows amino acid dependent expressionof sfGFP-Hiss bearing an amber codon at position 150. The expressedprotein was detected in lysates using an anti-Hiss antibody. Usingpurified exo or endo diastereomers of amino acid 1 demonstrated that theexo form is preferentially incorporated into sfGFP by BCNRS/tRNA_(CUA).

FIG. 18 (Supplementary Figure S7) shows LC-MS characterization of thelabelling reaction of sfGFP-1 with various tetrazines. Black peaksdenote the found mass of sfGFP-1 before labelling, colored peaks thefound masses after reaction of sfGFP-1 with 6, 7, 9 and 8. All massesare given in Daltons. Labelling with all tetrazines is specific andquantitative. Reaction conditions: to a ˜10 □M solution of sfGFP-1 (in20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 10 equivalents of thecorresponding tetrazine (1 mM stock solution in methanol) were added andthe reaction mixture incubated for 10 to 30 minutes at room temperature.

FIG. 19 (Supplementary Figure S8) shows LC-MS shows specific andquantitative labelling of sfGFP-1 with tetrazine fluorophore conjugates12, 16, 13 and 14. Red peaks denote the found mass of sfGFP-1 beforelabelling, colored peaks the found masses after reaction of sfGFP-1 with12(a), 16(b), 13(c) and 14(d). Expected and found mass values are givenin Daltons. Labelling with all tetrazine-fluorophores is specific andquantitative. Reaction conditions: to a ˜10 □M solution of sfGFP-1 (in20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4) 10 equivalents of thecorresponding tetrazine dye (2 mM stock solution in DMSO) were added andthe reaction mixture incubated for 10 to 30 minutes at room temperature.

FIG. 20 (Supplementary Figure S9) shows specificity of labeling 1 and 2in stGFP versus the E. coli proteome. The coomassie stained gel showsproteins from E. coli producing sfGFP in the presence of the indicatedconcentration of unnatural amino acids 1, 2, 3 (both exo and endodiastereomers) and 5. In gel fluorescence gels show specific labelingwith tetrazine-dye conjugate 11. Though amino acids 1, 2 and 3-exo areincorporated at a similar level (as judged from coomassie stained gelsand western blots), we observe only very faint, sub-stoichiometriclabeling of sfGFP produced in the presence of 3-exo and 3-endo. Theseobservations are consistent with the in vivo conversion of a fraction ofthe trans-alkene in 3 to its cis-isomer.

FIG. 21 (Supplementary Figure S10) shows specificity of labeling 1 insfGFP versus the E. coli proteome. Lanes 1-5: Coomassie stained gelshowing proteins from E. coli producing sfGFP in the presence of theindicated concentration of unnatural amino acids 1 and 5. Lanes 6-10:The expressed protein was detected in lysates using an anti-His6antibody. Lanes 11-15: fluorescence images of protein labeled with theindicated fluorophore 11.

FIG. 22 (Supplementary Figure S11) shows specific and ultra-rapidlabelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2minutes. EGFR-GFP bearing 1 at position 128 is visible as greenfluorescence at the membrane of transfected cells (left panels).Treatments of cells with 11 (400 nM) leads to selective labelling ofEGFR-GFP containing 1 (middle panels). Right panels show merged greenand red fluorescence images, DIC=differential interference contrast.Cells were imaged 2 minutes after addition of 11. No labelling wasobserved for cells in the same sample that did not express EGFR-GFP, andcells bearing EGFR-5-GFP were not labeled with 11.

FIG. 23 (Supplementary Figure S12) shows specific and ultra-rapidlabelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5minutes. EGFR-GFP bearing 1 at position 128 is visible as greenfluorescence at the membrane of transfected cells (left panels).Treatments of cells with 11 (400 nM) leads to selective labelling ofEGFR-GFP containing 1 (middle panels). Right panels show merged greenand red fluorescence images, DIC=differential interference contrast.Cells were imaged 5 minutes after addition of 11. No labelling wasobserved for cells in the same sample that did not express EGFR-GFP, andcells bearing EGFR-5-GFP were not labeled with 11.

FIG. 24 (Supplementary Figure S13) shows specific and ultra-rapidlabelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 10minutes. EGFR-GFP bearing 1 at position 128 is visible as greenfluorescence at the membrane of transfected cells (left panels).Treatments of cells with 11 (400 nM) leads to selective labelling ofEGFR-GFP containing 1 (middle panels). Right panels show merged greenand red fluorescence images, DIC=differential interference contrast.Cells were imaged 10 minutes after addition of 11. No labelling wasobserved for cells in the same sample that did not express EGFR-GFP, andcells bearing EGFR-5-GFP were not labeled with 11.

FIG. 25 (Supplementary Figure S14) shows that in contrast to theultra-rapid labelling of EGFR-GFP containing amino acid 1, it took 2hours to specifically label cells bearing EGFR-4-GFP withtetrazine-fluorophore conjugate 11.²

EGFR-GFP bearing 4 at position 128 is visible as green fluorescence atthe membrane of transfected cells (left panels). Treatments of cellswith 11 (200 nM) leads to labelling of EGFR-GFP containing 4 (middlepanels). Right panels show merged green and red fluorescence images,DIC=differential interference contrast. Cells were imaged 2 hours afteraddition of 11.

FIG. 26 (Supplementary Figure S15) shows specific and ultra-rapidlabelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 2minutes. EGFR-GFP bearing 2 at position 128 is visible as greenfluorescence at the membrane of transfected cells (left panels).Treatments of cells with 11 (400 nM) leads to selective labelling ofEGFR-GFP containing 2 (middle panels). Right panels show merged greenand red fluorescence images, DIC=differential interference contrast.Cells were imaged 2 minutes after addition of 11. No labelling wasobserved for cells in the same sample that did not express EGFR-GFP, andcells bearing EGFR-5-GFP were not labeled with 11.

FIG. 27 (Supplementary Figure S16) shows specific and ultra-rapidlabelling of EGFR-GFP with tetrazine-fluorophore conjugate 11 for 5minutes. EGFR-GFP bearing 2 at position 128 is visible as greenfluorescence at the membrane of transfected cells (left panels).Treatments of cells with 11 (400 nM) leads to selective labelling ofEGFR-GFP containing 2 (middle panels). Right panels show merged greenand red fluorescence images, DIC=differential interference contrast.Cells were imaged 5 minutes after addition of 11. No labelling wasobserved for cells in the same sample that did not express EGFR-GFP, andcells bearing EGFR-5-GFP were not labeled with 11.

FIG. 28 (Supplementary Figure S17) shows site specific incorporation of3 in mammalian cells and the labeling of EGFR-GFP withtetrazine-fluorophore conjugate 11 for 30 and 60 minutes. a) Westernblots demonstrate that the expression of full length mCherry(TAG)eGFP-HAis dependent on the presence of 3 or 5 and tRNA_(CUA). BCNRS and PylRSare FLAG tagged. B and c) EGFR-GFP in the presence 3 at position 128 isvisible as green fluorescence at the membrane of transfected cells (leftpanels). Treatments of cells with 11 (400 nM) leads to faint, butmeasurable labelling of EGFR-GFP containing 3 (middle panels) Thisobservation is consistent with the Isomerization of the trans-alkenebond to its cis form of a fraction of 3 in mammalian cells. Right panelsshow merged green and red fluorescence images, DIC=differentialinterference contrast. Cells were imaged 30 or 60 minutes after additionof 11. No labelling was observed for cells in the same sample that didnot express EGFR-GFP.

FIG. 29 (Supplementary Figure S18) shows specific and ultra-rapidlabelling of a nuclear protein in live mammalian cells. Jun-1-mCherry isvisible as red fluorescence in the nuclei of transfected cells (leftpanels). Treatment of cells with the cell permeable tetrazine dye 17(200 nM) leads to selective labeling of jun-1-mCherry (middle panel).Right panels show merged red and green fluorescence. DIC=differentialinterference contrast. Cells were imaged 15 minutes after addition of11. No labelling was observed for cells in the some sample that did notexpress jun-mCherry, and cells bearing jun-5-mCherry were not labeledwith 11

The invention is now described by way of example. These examples areintended to be illustrative, and are not intended to limit the appendedclaims.

EXAMPLES

Here we develop a rapid and fluorogenic reaction between tetrazines andBCN and demonstrate the genetic encoding of both BCN andtranscyclooctene containing amino acids 1 and 2 in E. coli and mammaliancells. We show the specific and rapid labeling of proteins in E. coliand in live mammalian cells with tetrazine probes, and explicitlydemonstrate the advantages of the approach with respect to previouslyreported bioorthogonal labeling strategies (FIG. 11—Scheme 1).

Example 1 Chemistry and Addition Reactions

The rate constants for the reactions of various dienophiles (BCN, TCO(trans-cyclooctene-4-ol) and sTCO(bicyclo[6.1.0]non-4-ene-9-ylmethanol)) with tetrazines have beendetermined^(3-5,9,11). However, in many cases, researchers have useddifferent tetrazines, solvent systems or measurement methods making itchallenging to quantitatively compare the reactivity of each dienophilewith tetrazines of interest. Our initial experiments confirmed that therates for the reactions of each dienophile with tetrazine 6 (FIG. 1)were too fast to study by manual mixing under pseudo first orderconditions. We therefore turned to stopped-flow techniques to directlydetermine the pseudo first order rate constants for these reactions. Byfollowing the exponential decay in absorbance at 320 nm upon reactionwith a 10- to 100-fold excess of BCN in a methanol/water (55/45) mixturewe determined the rate constants for the reaction of BCN with 6 and 7 as437 M⁻¹ s⁻¹ (+/−13) and 1245 M⁻¹ s⁻¹ (+/−45), respectively. LC-MS andNMR confirm the formation of the expected products (SupplementaryInformation and Supplementary FIG. 1). Under the same conditions wedetermined the rate constant of TCO with 6 and 7 as 5235 M⁻¹ s⁻¹(+/−258) and 17248 M⁻¹ s⁻¹ (+/−3132), respectively. These datademonstrate that the reaction between BCN and 6 is approximately 1000times faster than the reaction between 5-norbornene-2-ol and 6⁷, whilethe TCO rate is approximately 10-15 times faster than the BCN rate. ThesTCO rate was too fast to be measured accurately by stopped flowtechniques and we estimate that it is at least 50 times faster than theTCO rate. Similar rate accelerations were observed for the reaction ofBCN with tetrazines 8 and 9 (FIG. 1, FIGS. 2 a and 2 b, SupplementaryTable 1 and Supplementary Figures S2 and S3).

SUPPLEMENTARY TABLE 1 Tetrazine BCN k₂ [M⁻¹s⁻¹]^(a) Nor k₂ [M⁻¹s⁻¹]^(a)TCO k₂ [M⁻¹s⁻¹]^(a) 6 437 ± 13 0.47 ± 0.0069 5235 ± 258 7 1245 ± 45 1.70 ± 0.048  17248 ± 3132 9 80 0.15 n.d. 8 2672 ± 95  5.00 ± 0.096 n.d. Rate constants k for the reaction of various tetrazines (6, 7, 9and 8) with BCN and TCO at 25° C. measured under pseudo first orderconditions using a stopped-flow device in comparison to rate constantsfor the reaction of the same tetrazines with 5-norborriene-2-ol at 21°C.² Values were determined from at least two independent measurements.Solvent system: 55/45 methanol/water. The cycloaddition reaction of BCNto tetrazines is 500 to 1000 times faster than the one of5-norbornene-2-ol, the reaction between TCO and tetrazines is 10 to 15times faster than the one between BCN and tetrazines.

Several tetrazine fluorophore conjugates, including 11, 13, 14 and 16(FIG. 1, Supplementary Figure S4) are substantially quenched withrespect to the free fluorophore, an observation that results from energytransfer of the fluorophore's emission to a proximal tetrazinechromophore with an absorption maximum between 510 and 530 nm^(7,18). Wefind that the reaction of BCN with tetrazine fluorophore conjugates 11,13, 14 and 16 leads to a 5-10 fold increase in fluorescence, suggestingthat the formation of the pyridazine product efficiently relievesfluorophore quenching (FIG. 2 c and Supplementary Figure S5). Thefluorogenic reaction between BCN and these tetrazines, like the reactionbetween strained alkenes and these tetrazines^(7,18), is advantageousfor imaging experiments since it maximizes the labeling signal whileminimizing fluorescence arising from the free tetrazine fluorophore.

Example 2 Amino Acid Design

Next, we aimed to design, synthesize and genetically encode amino acidsbearing BCN. TCO and sTCO for site-specific protein labeling with adiverse range of probes both in vitro and in cells. The Pyrrolysyl-tRNAsynthetase (PylRS)/tRNA_(CUA) pairs from Methanosarcina species,including M. barkeri (Mb) and M. mazei (Mm), and their evolvedderivatives have been used to direct the site-specific incorporation ofa growing list of structurally diverse unnatural amino acids in responseto the amber codon¹⁹⁻²⁶. The PylRS/tRNA_(CUA) pair is emerging asperhaps the most versatile system for incorporating unnatural aminoacids into proteins since it is orthogonal in a range of hosts, allowingsynthetases evolved in E. coli to be used for genetic code expansion ina growing list of cells and organisms, including: E. coli, Salmonellatyphimurium, yeast, human cells and C. elegans ^(7,27-31). We designedthe unnatural amino acids 1, 2 and 3 (FIG. 1) with the goal ofincorporating them into proteins using the PylRS/tRNA_(CUA) pair or anevolved derivative. The amino acids were synthesized as described in theSupplementary Information.

Example 3 Genetic Incorporation into Polypeptides and tRNA Synthetases

We screened the MbPylRS/tRNA_(CUA) pair along with a panel of mutants ofMbPylRS, previously generated in our laboratory for the site-specificincorporation of diverse unnatural amino acids into proteins, for theirability to direct the incorporation of 1, 2 and 3 in response to anamber codon introduced at position 150 in a C-terminally hexahistidine-(His₆) tagged superfolder green fluorescent protein (sfGFP). TheMbPylRS/tRNA_(CUA) pair did not direct the incorporation of any of theunnatural amino acids tested, as judged by western blot against theC-terminal His₆ tag. However, cells containing a mutant of MbPylRS,containing three amino acid substitutions Y271M, L274G, C313A³² in theenzyme active site (which we named BCN-tRNA synthetase, BCNRS), and aplasmid that encodes MbtRNA_(CUA) and sfGFP-His₆ with an amber codon atposition 150 (psfGFP150TAGPylT-His₆) led to amino acid dependentsynthesis of full length sfGFP-His₆, as judged by anti-Hiss western blotand coomassie staining (FIG. 3 a). Additional protein expressionexperiments using 1, and its endo isomer demonstrated that the exo formis preferentially incorporated into proteins by BCNRS/tRNA_(CUA)(Supplementary Figure S6). We found an additional synthetase mutant,bearing the mutations Y271A, L274M and C313A³², which we named TCO-tRNAsynthetase, TCORS. The TCORS/tRNA_(CUA) pair led to amino acid dependentsynthesis of sfGFP from psfGFP150TAGPylT-His₆ in the presence of 2.Finally we found that both the BCNRS/tRNA_(CUA) pair as well as theTCORS/tRNA_(CUA) pair led to amino acid dependent synthesis of sfGFPfrom psfGFP150TAGPylT-His₆ in the presence of 3. For each amino acidsfGFP was isolated in good yield after His-tag and gel filtrationpurification (6-12 mg per L of culture. FIG. 3 b). This is comparable tothe yields obtained for other well-incorporated unnatural amino acids,including 5. Electrospray ionization mass spectrometry (ESI-MS) of sfGFPproduced from psfGFP150TAGPylT-His₆ in the presence of each unnaturalamino acid is consistent with their site-specific incorporation (FIG. 3c-3 e).

Example 4 Site-Specific Incorporation

To demonstrate that the tetrazine-dye-probes react efficiently andspecifically with recombinant proteins that bear site-specificallyincorporated 1 we labeled purified sfGFP-1-His₆ with 10 equivalents oftetrazine fluorophore conjugate 11 for 1 hour at room temperature.SDS-page and ESI-MS analysis confirmed quantitative labeling of sfGFPcontaining 1 (FIGS. 4 a and 4 b). Control experiments demonstrated thatsfGFP-4 is labeled under the same conditions used to label sfGFP-1, andthat no non-specific labeling is detected with sfGFP-5. ESI-MSdemonstrates that sfGFP-1 can be efficiently and specificallyderivatized with a range of tetrazines 6, 7, 8 and 9 (SupplementaryFigure S7), and with tetrazine fluorophore conjugates 12, 13, 14 and 16(Supplementary Figure S8). We also demonstrated that purifiedsfGFP-2-His₆ can be quantitatively labeled with tetrazine fluorophore 11(FIGS. 4 a and 4 c). Interestingly we observe only very faint labelingof sfGFP-Hiss purified from cells expressing the TCORS/tRNA_(CUA) andpsfGFP150TAGPylT-His₆ and grown in the presence of 3 (FIGS. 4 a and 4 d)and sub-stoichiometric labeling of this protein prior to purification(Supplementary Figure S9). Since the sfGFP expressed in the presence of3 has a mass corresponding to the incorporation of 3, these observationsare consistent with the in vivo conversion of a fraction of thetrans-alkene in 3 to its unreactive cis isomer. This isomerization isknown to occur in the presence of thiols.⁴

Example 5 Specificity and Selectivity of Reactions

To further demonstrate that the reaction between BCN and varioustetrazine-based dyes is not only highly efficient and specific, but alsohighly selective within a cellular context, we performed the reaction onE. coli expressing sfGFP-1-His₆ (Supplementary Figure S10). Cellsexpressing sfGFP-1 at a range of levels (controlled by adjusting theconcentration of 1 added to cells) were harvested 4 hours afterinduction of protein expression, washed with PBS and incubated withtetrazine dye 11 for 30 min at room temperature. After adding an excessof BCN in order to quench non-reacted tetrazine-dye, the cells werelysed and the reaction mixtures were analyzed. In-gel fluorescencedemonstrated specific labeling of recombinant sfGFP bearing 1 withtetrazine-conjugated TAMRA dye 11. While many proteins in the lysateswere present at a comparable abundance to sfGFP-1 we observe very littlebackground labeling, suggesting that the reaction is specific withrespect to the E. coli proteome.

Example 6 Speed of Labelling

To investigate whether the rate of reaction for the BCN- andTCO-tetrazine cycloadditions observed on small molecules translates intoexceptionally rapid protein labeling we compared the labeling ofpurified sfGFP bearing 1, 2 or 4 with 10 equivalents oftetrazine-fluorophore conjugate 11. In-gel fluorescence imaging of thelabeling reaction as a function of time (FIG. 4 e) indicates that thereaction of sfGFP-4 reaches completion in approximately 1 h. In contrastthe labeling of sfGFP-1 and sfGFP-2 was complete within the few secondsit took to measure the first time point, demonstrating that the rateacceleration of the BCN- and TCO-tetrazine reaction translates into muchmore rapid protein labeling.

Example 7 Application to Mammalian Cells

To demonstrate the incorporation of amino acids 1 and 2 in mammaliancells we created mammalian optimized versions of BCNRS and TCORS bytransplanting the mutations that allow the incorporation of 1 or 2 intoa mammalian optimized MbPylRS. By western blot we demonstrated that both1 and 2 can be genetically encoded with high efficiency into proteins inmammalian cells using the BCNRS/tRNA_(CUA) pair or TCORS/tRNA_(CUA)(FIG. 5 a).

To investigate whether the rapid BCN-tetrazine ligation providesadvantages for site-specifically labeling proteins on mammalian cells weexpressed an epidermal growth factor receptor (EGFR)—green fluorescentprotein (GFP) fusion bearing an amber codon at position 128(EGFR(128TAG)GFP) in HEK-293 cells containing the BCNRS/tRNA_(CUA) pair,cultured in the presence of 1 (0.5 mM). Full-length EGFR-1-GFP wasproduced in the presence of 1 resulting in bright green fluorescence atthe cell membrane. To label 1 at position 128 of EGFR, which is on theextracellular domain of the receptor, with tetrazine-fluorophoreconjugates we incubated cells with 11 (400 nM), changed the media andimaged the red fluorescence arising from TAMRA labeling as well as thegreen fluorescence arising from expression of full-length EGFR-GFP,TAMRA fluorescence co-localized nicely with cell-surface EGFR-GFPfluorescence. Clear labeling of cells that bear EGFR-1-GFP was observedwithin 2 minutes, the first time point we could measure; additional timepoints demonstrated that labeling was saturated within 2 minutes (FIG. 5b and Supplementary Figures S11 -S14); similar results were obtainedwith tetrazine fluorophore 12. Incorporation of 2 into the EGFR-GFPfusion led to similarly rapid and efficient labeling with tetrazinefluorophore 11 (FIG. 5 b and Supplementary Figures S15-S16). In contrastit took 2 hours before we observed any specific labeling of cellsbearing EGFR-4-GFP under identical conditions (Supplementary FigureS14)⁷. In control experiments we observed no labeling for cells bearingEGFR-5-GFP and no non-specific labeling was detected for cells that didnot express EGFR-GFP. We observe weak but measurable labeling ofEGFR-GFP expressed in HEK 293 cells from (EGFR(128TAG)GFP) in thepresence of the BCNRS/tRNA_(CUA) pair and 3 (Supplementary Figure S17).These observations are consistent with the isomerization of a fractionof 3 in mammalian cells, and with our observations in E. coli.

To demonstrate the rapid labeling of an intracellular protein inmammalian cells we expressed a transcription factor, jun, with aC-terminal mCherry fusion from a gene bearing an amber codon in thelinker between JunB (jun) and mCherry. In the presence of amino acid 1and the BCNKRS/tRNA_(CUA) pair the jun-1-mCherry protein was produced inHEK cells and, as expected, localized to the nuclei of cells (FIG. 5 cand Supplementary Figure S18). Labeling with a cell permeable diacetylfluorescein tetrazine conjugate (200 nM) resulted in green fluorescencethat co-localizes nicely with the mCherry signal at the first time pointanalyzed (15 min labeling followed by 90 min washing). No specificlabeling was observed in non-transfected cells in the same sample or incontrol cells expressing jun-5-mCherry, further confirming thespecificity of intracellular labeling.

Supplementary Examples Protein Expression and Purification

To express sfGFP with incorporated unnatural amino acid 1, wetransformed E. coli DH10B cells with pBKBCNRS (which encodes MbBCNRS)and psfGFP150TAGPylT-His₆ (which encodes MbtRNA_(CUA) and a C-terminallyhexahistidine tagged sfGFP gene with an amber codon at position 150).Cells were recovered in 1 ml of S.O.B media (supplemented with 0.2%glucose) for 1 h at 37° C., before incubation (16 h, 37° C., 230 r.p.m)in 100 ml of LB containing ampicillin (100 μg/mL) and tetracycline (25μg/mL). 20 ml of this overnight culture was used to inoculate 1 L of LBsupplemented with ampicillin (50 μg/mL) and tetracycline (12 μg/mL) andincubated at 37° C. At OD₆₀₀=0.4 to 0.5, a solution of 1 in H₂O wasadded to a final concentration of 2 mM. After 30 min, protein expressionwas induced by the addition of arabinose to a final concentration of0.2%. After 3 h of induction, cells were harvested by centrifugation andfrozen at −80° C. until required. Cells were thawed on ice and suspendedin 30 ml of lysis buffer (10 mM Tris-HCl, 20 mM imidazole, 200 mM NaCl,pH 8, 1 mM phenylmethanesulfonylfluoride, 1 mg/mL lysozyme, 100 μg/mLDNaseA, Roche protease inhibitor). Proteins were extracted by sonicationat 4° C. The extract was clarified by centrifugation (20 min, 21.000 g,4° C.), 600 μL of Ni²⁺-NTA beads (Qiagen) were added to the extract andthe mixture was incubated with agitation for 1 h at 4° C. Beads werecollected by centrifugation (10 min, 1000 g). The beads were three timesresuspended in 30 mL wash buffer (20 mM Tris-HCl, 30 mM imidazole, 300mM NaCl, pH 8) and spun down at 1000 g. Subsequently, the beads wereresuspended in 10 mL of wash buffer and transferred to a column. Theprotein was eluted with 3 ml of wash buffer supplemented with 200 mMimidazole and further purified by size-exclusion chromatographyemploying a HiLoad 16/60 Superdex 75 Prep Grade column (GE LifeSciences) at a flow rate of 1 mL/min (buffer: 20 mM Tris-HCl, 100 mMNaCl, 2 mM EDTA, pH 7.4). Fractions containing the protein were pooledand concentrated with an Amicon Ultra-15 3 kDa MWCO centrifugal filterdevice (Millipore). Purified proteins were analyzed by 4-12% SDS-PAGEand their mass confirmed by mass spectrometry (see SupplementaryInformation). SfGFP with incorporated 2 and 3, sfGFP-2, sfGFP-3 wereprepared in the same way, expect that cells were transformed withpBKTCORS (which encodes MbTCORS) and psfGFP150TAGPylT-His₆ (whichencodes MbtRNA_(CUA) and a C-terminally hexahistidine tagged sfGFP genewith an amber codon at position 150). SfGFP with incorporated 4 and 5,sfGFP-4, sfGFP-5 were prepared in the same way, expect that cells weretransformed with pBKPylRS (which encodes MbPylRS) andpsfGFP150TAGPylT-His₆ (which encodes MbtRNA_(CUA) and a C-terminallyhexahistidine tagged sfGFP gene with an amber codon at position 150).Yields of purified proteins were up to 6-12 mg/L.

Protein Mass Spectrometry

Using an Agilent 1200 LC-MS system, ESI-MS was carried out with a 6130Quadrupole spectrometer. The solvent system consisted of 0.2% formicacid in H₂O as buffer A, and 0.2% formic acid in acetonitrile (MeCN) asbuffer B. LC-ESI-MS on proteins was carried out using a PhenomenexJupiter C4 column (150×2 mm, 5 μm) and samples were analyzed in thepositive mode, following protein UV absorbance at 214 and 280 nm. Totalprotein masses were calculated by deconvolution within the MSChemstation software (Agilent Technologies).

Additionally, protein total mass was determined on an LCT time-of-flightmass spectrometer with electrospray ionization (ESI, Micromass).Proteins were rebuffered in 20 mM of ammonium bicarbonate and mixed 1:1acetonitrile, containing 1% formic acid. Alternatively samples wereprepared with a C4 Ziptip (Millipore) and infused directly in 50%aqueous acetonitrile containing 1% formic acid. Samples were injected at10 μL min⁻¹ and calibration was performed in positive ion mode usinghorse heart myoglobin. 30 scans were averaged and molecular massesobtained by maximum entropy deconvolution with MassLynx version 4.1(Micromass). Theoretical masses of wild-type proteins were calculatedusing Protparam (http://us.expasy.org/tools/protparam.html), andtheoretical masses for unnatural amino acid containing proteins wereadjusted manually.

Protein Labelling Via Tetrazine-BCN or Tetrazine-TCO Cycloaddition

In Vitro Labelling of Purified Proteins with Different Tetrazines

To 40 μL of purified recombinant protein (˜10 μM in 20 mM Tris-HCl, 100mM NaCl, 2 mM EDTA, pH 7.4) 4 μL of a 1 mM solution of tetrazinecompounds 6, 7, 8, or 9 in MeOH were added (˜10 or 20 equivalents).After 30 minutes of incubation at room temperature, the solutions wereanalyzed by LC-ESI-MS. (Supplementary Figure S9)

In Vitro Labelling of Purified Proteins with Tetrazines andTetrazine-Dye Conjugates:

Purified recombinant sfGFP with site-specifically incorporated 1 or 2,sfGFP-1 or sfGFP-2 (˜10 μM in 20 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH7.4), was incubated with 10 equivalents of the tetrazine-dye conjugates11, 12, 13, 14, 15 or 16, respectively (2 mM in DMSO). The solution wasincubated at room temperature and aliquots were taken after 30 min to 3hours and analyzed by SDS PAGE and —after desalting with a C4-ZIPTIP—byESI-MS. The SDS PAGE gels were either stained with coomassie or scannedwith a Typhoon imager to visualize in-gel fluorescence (FIG. 4 andSupplementary Figure S8).

In Vitro Labelling of Purified Proteins with Tetrazines-Dye Conjugatesas a Function of Time:

2 nmol of purified sfGFP-1, sfGFP-2 or sfGFP-4 (10 μM in 20 mM Tris-HCl,100 mM NaCl, 2 mM EDTA, pH 7.4) were incubated with 20 nmol oftetrazine-dye conjugate 11 (10 μl of a 2 mM solution in DMSO). Atdifferent time points (0, 30 s, 1 min, 2 min, 5 min, 10 min, 30 min, 1h, 2 h, 3 h) 8 μL aliquots were taken from the solution and quenchedwith a 700-fold excess of BCN or TCO and plunged into liquid nitrogen.Samples were mixed with NuPAGE LDS sample buffer supplemented with 5%β-mercaptoethanol, heated for 10 min to 90° C. and analyzed by 4-12% SDSpage. The amounts of labelled proteins were quantified by scanning thefluorescent bands with a Typhoon Trio phosphoimager (GE Life Sciences).Bands were quantified with the ImageQuant™ TL software (GE LifeSciences) using rubber band background subtraction. In gel fluorescenceshows that labelling is complete within 1 h for sfGFP-4 using 10equivalents tetrazine-fluorophore 11 (FIG. 4 e), whereas the labellingof sfGFP-1 and sfGFP-2 was complete within the few seconds it took tomeasure the first time point.

Labelling of the Whole E. coli Proteome with Tetrazine-Dye Conjugates:

E. coli DH10B cells containing either psfGFP150TAGPylT-His₆ and pBKBCNRSor psfGFP150TAGPylT-His₆ and pBKPylRS were inoculated into LB containingampicillin (for pBKBCNRS, 100 μg/mL) or kanamycin (for pBKPylRS 50μg/mL) and tetracycline (25 μg/mL). The cells were incubated withshaking overnight at 37° C., 250 rpm. 2 mL of overnight culture was usedto inoculate into 100 mL of LB supplemented with ampicillin (50 μg/mL)and tetracycline (12 μg/mL) or kanamycin (25 μg/mL) and tetracycline (12μg/mL) and incubated at 37° C. At OD₆₀₀=0.5, 3 ml culture aliquots wereremoved and supplemented with different concentrations (1 mM, 2 mM and 5mM) of 1 and 1 mM of 5. After 30 min of incubation with shaking at 37°C., protein expression was induced by the addition of 30 μL of 20%arabinose. After 3.5 h of expression, cells were collected bycentrifugation (16000 g, 5 min) of 1 mL of cell suspension. The cellswere resuspended in PBS buffer, spun down again and the supernatant wasdiscarded. This process was repeated twice more. Finally, the washedcell pellet was suspended in 100 μL PBS and incubated with 3 μL oftetrazine-dye conjugate 11 (2 mM in DMSO) at rt for 30 minutes. Afteradding a 200-fold excess of BCN in order to quench non-reactedtetrazine-dye, the cells were resuspended in 100 μL of NuPAGE LDS samplebuffer supplemented with 5% β-mercaptoethanol, heated at 90° C. for 10min and centrifuged at 16000 g for 10 min. The crude cell lysate wasanalyzed by 4-12% SDS-PAGE to assess protein levels. Gels were eitherCoomassie stained or scanned with a Typhoon imager to make fluorescentbands visible (Supplementary Figures S9 and S10). Western blots wereperformed with antibodies against the hexahistidine tag (Cell SignalingTechnology, His tag 27E8 mouse mAb #2366).

Stopped-Flow Determination of Kinetic Rate Constants for Small MoleculeCycloadditions

Rate constants k for different tetrazines were measured under pseudofirst order conditions with a 10- to 100-fold excess of BCN or TCO inmethanol/water mixtures by following the exponential decay in UVabsorbance of the tetrazine at 320, 300 or 280 nm over time with astopped-flow device (Applied Photophysics, Supplementary Figures S2 andS3 and Supplementary Table 1). Stock solutions were prepared for eachtetrazine (0.1 mM in 9/1 water/methanol) and for BCN and TCO (1 to 10 mMin methanol). Both tetrazine and BCN and TCO solutions werethermostatted in the syringes of the stopped flow device beforemeasuring. Mixing equal volumes of the prepared stock solutions via thestopped-flow apparatus resulted in a final concentration of 0.05 mMtetrazine and of 0.5 to 5 mM BCN or TCO, corresponding to 10 to 100equivalents of BCN or TCO. Spectra were recorded using the followinginstrumental parameters: wavelength, 320 nm for 6 and 7; 300 nm for 8,280 nm for 9; 500 to 5000 datapoints per second). All measurements wereconducted at 25° C. Data were fit to a single-exponential equation forBCN-tetrazine reactions and to a sum of two single exponential equationsfor TCO-tetrazine reactions. Each measurement was carried out three tofive times and the mean of the observed rates k′ (the first exponentialequation in case of the TCO-tetrazine reaction) was plotted against theconcentration of BCN or TCO to obtain the rate constant k from the slopeof the plot. For all four tetrazines complete measurement sets were donein duplicate and the mean of values is reported in SupplementaryTable 1. All data processing was performed using Kaleidagraph software(Synergy Software, Reading, UK).

Cloning for Mammalian Cell Applications

The plasmids pMmPylS-mCherry-TAG-EGFP-HA^(1,2) andpMmPylRS-EGFR-(128TAG)-GFP-HA² were both digested with the enzymes AflIIand EcoRV (NEB) to remove the wild-type MmPylRS. A synthetic gene of themutant synthetase MbBCNRS and MbTCORS was made by GeneArt with the sameflanking sites. The synthetic MbBCNRS and MbTCORS were also digestedwith AflII and EcoRV and cloned in place of the wild-type synthetase(MmPylS). Using a rapid ligation kit (Roche) vectorspMbBCNRS-mCherry-TAG-EGFP-HA, pMbBCNRS-EGFR(128TAG)-GFP-HA andpMbTCORS-EGFR(128TAG)-GFP-HA were created. ThepCMV-cJun-TAG-mCherry-MbBCNRS plasmid was created from apCMV-cJun-TAG-mCherry-MmPylRS plasmid (created by Fiona Townsley) byexchanging MmPylRS for MbBCNRS. This was carried out as for thepMbBCNRS-mCherry-TAG-EGFP-HA plasmid.

Incorporation of Amino Acid 1, 2 and 3 in HEK293 Cells

HEK293 cells were plated on poly-lysine coated μ-dishes (Ibidi). Aftergrowing to near confluence in 10% fetal bovine serum (FBS) Dulbecco'smodified eagle medium (DMEM) cells were transfected with 2 μg ofpMbBCNRS-EGFR(128TAG)-GFP-HA and 2 μg of p4CMVE-U6-PylT (which containsfour copies of the wild-type pyrrolysyl tRNA)^(1,2) using lipofectamin2000 (Life Technologies). After transfection cells were left to growovernight in 10% FBS DMEM at 37° C. and 5% CO₂. For a western blot,cells were plated on 24 well plates and grown to near confluence. Cellswere transfected using lipofectamine 2000 with thepMbBCNRS-mCherry-TAG-EGFP-HA or pMmPylRS-mCherry-TAG-EGFP-HA orpTCORS-mCherry-TAG-EGFP-HA construct and the p4CMVE-U6-PylT plasmid.After 16 hours growth with or without 0.5 mM 1, 1 mM 2 or 1 mM 5 cellswere lysed on ice using RIPA buffer (Sigma). The lysates were spun downand the supernatant was added to 4×LDS sample buffer (Lifetechnologies). The samples were run out by SDS-PAGE, transferred to anitrocellulose membrane and blotted using primary rat anti-HA (Roche)and mouse anti-FLAG (Ab frontier), secondary antibodies were anti-rat(Santa Cruz Biotech) and anti-mouse (Cell Signaling) respectively.

Labelling of Mammalian Cell Surface Protein

Cells were plated onto a poly-lysine coated μ-dish and after growing tonear confluence were transfected with 2 μg each ofpMbBCNRS-EGFR(128TAG)-GFP-HA or pMbTCORS-EGFR(128TAG)-GFP-HA andp4CMVE-U6-PylT. After 8-16 hours growth at 37° C. and at 5% CO₂ in DMEMwith 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO), 1 mM 2 or 1 mM 3cells were washed in DMEM with 0.1% FBS and then incubated in DMEM with0.1% FBS overnight. The following day cells were washed once more before400 nM terazine-dye conjugate 11 was added for 2-60 minutes. The mediawas exchanged twice and cells were then imaged. Imaging was carried outon a Zeiss 780 laser scanning microscope with a Plan apochromat 63× oilimmersion objective; scan zoom: 1× or 2×; scan resolution: 512×512; scanspeed: 9; averaging: 16×. EGFP was excited at 488 nm and imaged at 493to 554 nm; TAMRA was excited and detected at 561 nm and 566-685 nmrespectively.

Controls were performed similarly but transfected withpMmPylRS-EGFR(128TAG)-GFP-HA instead of pMbBCNRS-EGFR(128TAG)-GFP-HA.Cells were grown overnight in the presence of 1 mM 5 and in the absenceor presence of 0.5% DMSO (as would be the case for amino acid 1).

Labeling of Mammalian Nuclear Protein

Cells were plated onto a poly-lysine coated μ-dish and after growing tonear confluence were transfected with 2 μg each of pCMV-cJun-TAG-mCherryand p4CMVE-U6-PylT. After approximately 16 hrs growth at 37° C. and at5% CO₂ in DMEM with 0.1% FBS in the presence of 0.5 mM 1 (0.5% DMSO)cells were washed in DMEM 0.1% FBS and then incubated in DMEM 0.1% FBSovernight. The following day cells were washed repeatedly, using twomedia exchanges followed by 30 minutes incubation over 2 hours. 200 nMtetrazine-dye conjugate 11 was added for 15 minutes, the cells were thenrepeatedly washed again for 90 mins. Imaging was carried out as for thecell surface labeling

Chemical Syntheses: General Methods:

NMR spectra were recorded on a Bruker Ultrashield™ 400 Plus spectrometer(¹H: 400 MHz, ¹³C: 101 MHz, ³¹P: 162 MHz). Chemical shifts (δ) arereported in ppm and are referenced to the residual non-deuteratedsolvent peak: CDCl₃ (7.26 ppm), d₆-DMSO (2.50 ppm) for ¹H-NMR spectra,CDCl₃ (77.0 ppm), d₆-DMSO (39.5 ppm) for ¹³C-NMR spectra. ¹³C- and³¹P-NMR resonances are proton decoupled. Coupling constants (J) aremeasured to the nearest 0.1 Hz and are presented as observed. Splittingpatterns are designated as follows: s, singlet; d, doublet; t, triplet;q, quartet; quin, quintet; sext, sextet; m, multiplet. Analyticalthin-layer chromatography (TLC) was carried out on silica 60F-254plates. The spots were visualized by UV light (254 nm) and/or bypotassium permanganate staining. Flash column chromatography was carriedout on silica gel 60 (230-400 mesh or 70-230 mesh). ESI-MS was carriedout using an Agilent 1200 LC-MS system with a 6130 Quadrupolespectrometer. The solvent system consisted of 0.2% formic acid in H₂O asbuffer A, and 0.2% formic acid in acetonitrile (MeCN) as buffer B. Smallmolecule LC-MS was carried out using a Phenomenex Jupiter C18 column(150×2 mm, 5 m). Variable wavelengths were used and MS acquisitions werecarried out in positive and negative ion modes. Preparative HPLCpurification was carried out using a Varian PrepStar/ProStar HPLCsystem, with automated fraction collection from a Phenomenex C18 column(250×30 mm, 5 μm). Compounds were identified by UV absorbance at 191 nm.All solvents and chemical reagents were purchased from commercialsuppliers and used without further purification.Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN, exo/endo mixture ˜4/1) waspurchased from SynAffix, Netherlands. Non-aqueous reactions were carriedout in oven-dried glassware under an inert atmosphere of argon unlessstated otherwise. All water used experimentally was distilled. Brinerefers to a saturated solution of sodium chloride in water.

exo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) was synthesisedaccording to a literature procedure.³

N,N′-disuccinimidyl carbonate (1.38 g, 5.37 mmol) was added to astirring solution of exo-BCN-OH S18 (538 mg, 3.58 mmol) andtriethylamine (2.0 mL, 14.3 mmol) in MeCN (10 mL) at 0° C. The solutionwas warmed to room temperature and stirred for 3 h and concentratedunder reduced pressure. The crude oil was purified through a short padof silica gel chromatography (eluting with 60% EtOAc in hexane) to yieldthe exo-BCN-succinimidyl carbonate, which was used without furtherpurification. exo-BCN-OSu (1.25 g, 4.29 mmol) in DMF (4 mL) was addedvia cannula to a stirring solution of Fmoc-Lys-OH.HCl (2.61 g, 6.45mmol) and DIPEA (1.49 mL, 8.58 mmol) in DMF (10 mL). The solution wasstirred at room temperature for 14 h, diluted with Et₂O (100 mL) andwashed with H₂O (3×100 mL). The organic phase was dried over sodiumsulfate, filtered and concentrated under reduced pressure. The crude oilwas purified by silica gel chromatography (0-5% MeOH in DCM (0.1% AcOH))to yield exo-Fmoc-BCNK-OH S19 as a white solid (1.65 g, 85% over 2steps). δ_(H) (400 MHz, d₆-DMSO) 12.67-12.31 (1H, br s), 7.90 (2H, d, J7.5), 7.73 (2H, d, J 7.4), 7.63 (1H, d, J 7.8), 7.42 (2H, t, J 7.4),7.34 (2H, t, J 7.4), 7.10 (1H, t, J 5.7), 4.31-4.19 (3H, m), 3.95-3.87(1H, m), 3.84 (1H, d, J 6.4), 3.45-3.25 (br s, 1H), 3.01-2.91 (2H, m),2.52-2.50 (1H, m), 2.33-2.15 (4H, m), 2.11-2.02 (2H, m), 1.75-1.54 (2H,m), 1.46-1.23 (6H, m), 0.70-0.58 (2H, m); δ_(C) (101 MHz, d₆-DMSO)174.4, 156.9, 156.6, 144.30, 144.27, 141.2, 128.1, 127.5, 125.7, 120.6,99.4, 68.1, 66.1, 54.3, 47.1, 33.3, 30.9, 29.5, 23.9, 23.4, 22.7, 21.3;LRMS (ESI⁺): m/z 543 (100% [M−H]⁻).

Polymer-bound piperazine (1.28 g, 1.28 mmol, 200-400 mesh, extent oflabeling: 1.0-2.0 mmol/g loading, 2% cross-linked with divinylbenzene)was added to a stirring solution of exo-Fmoc-BCNK-OH S19 (174 mg, 0.32mmol) in DCM (10 mL). The resulting mixture was stirred for 4 h at roomtemperature, filtered and the reagent washed with CHCl₃/MeOH (3:1, 3×50mL). The filtrate was evaporated under reduced pressure, dissolved inH₂O (100 mL) and washed with EtOAc (3×100 mL). The aqueous phase wasevaporated under reduced pressure and freeze-dried to yieldexo-H-BCNK-OH 1 as a white solid (101 mg, 98%). For all subsequentlabeling experiments using mammalian cells exo-H-BCNK-OH 1 was furtherpurified by reverse-phase HPLC (0:1 H₂O:MeCN to 9:1 H₂O:MeCN gradient).δ_(H) (400 MHz, d₆-DMSO/D₂O (1:1)) 4.14-3.76 (m, 3H), 3.56-3.29 (m, 2H),3.18-2.81 (m, 3H), 2.31-1.98 (m, 5H), 1.71-1.52 (m, 4H), 1.51-1.29 (m,4H), 1.29-1.08 (m, 3H), 0.95-0.66 (m, 2H); δ_(C) (101 MHz, d₆-DMSO/D₂O(1:1)) 169.4, 165.9, 101.3, 76.0, 55.8, 31.8, 30.1, 29.9, 25.2, 23.2,22.1, 21.0, 18.7; LRMS (ESI⁺): m/z 323 (100% [M+H]⁺).endo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (endo-BCN) was synthesisedaccording to a literature procedure³ and elaborated to the correspondingamino acid in an analogous fashion to 1.

A glass vial (Biotage® Ltd.) equipped with a magnetic stirring bar wascharged with compound 6 (39.2 mg, 0.096 mmol) and was sealed with anair-tight aluminium/rubber septum. The contents in the vial were driedin vacuo and purged with argon gas (×3). MeOH (1 ml) was added to thevial, followed by addition of a solution ofexo-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (exo-BCN, S18) (20.2 mg in 1 mlof MeOH, 0.1344 mmol). The mixture was stirred at room temperature.Within 2 min, the reaction mixture decolorised and the contents wereleft stirring for additional 1 min. The mixture was then evaporatedunder reduced pressure and purified by silica gel chromatography (5%MeOH in DCM) to afford pyridazine S20 as a faint yellow semi-solid (49mg, 96%). δ_(H) (400 MHz, CDCl₃) 9.16 (1H, br s), 8.77-8.71 (1H, m),8.67 (1H, app. d, J 2.1), 8.01 (1H, br s), 7.97 (1H, d, J 7.8), 7.89(1H, ddd, J 7.8, 7.6, 1.7), 7.75 (1H, app. d, J 8.4), 7.40 (1H, ddd, J7.4, 4.9, 1.1), 5.93 (1H, br s), 4.02 (2H, d, J 5.0), 3.49-3.31 (2H, m),3.12-2.88 (4H, m), 2.68-2.49 (2H, m), 1.88-1.60 (1H, br s), 1.60-1.50(1H, m), 1.48 (9H, s), 0.92-0.72 (4H, m); δ_(C) (101 MHz, CDCl₃) 169.0,159.2, 159.0, 156.9, 156.8, 155.7, 152.1, 148.9, 143.0, 140.9, 137.0,134.4, 128.0, 125.1, 124.9, 123.5, 80.7, 66.4, 45.7, 30.7, 29.9, 29.6,29.5, 28.5 (3×CH₃ (^(t)Bu)), 28.0, 27.8, 21.7; LRMS (ESI⁺): m/z 531(100% [M+H]⁺).

Commercially available 4-(Aminomethyl)benzonitrile hydrochloride S21(2.11 g, 12.50 mmol) in H₂O (10 mL) was added to a stirring solution ofNaOH (1.50 g, 37.50 mmol) and di-tert-butyl dicarbonate (3.00 g, 13.75mmol) in H₂O (10 mL) at room temperature. The mixture was stirred for 16h, after which time a white precipitate had formed. The mixture wasfiltered, washed with H₂O (50 mL), and the resulting solid dried undervacuum to yield tert-butylcarbamate S22 as a white solid (2.78 g, 96%).δ_(H) (400 MHz, CDCl₃) 7.62 (2H, d, J 8.2), 7.39 (2H, d, J 8.2), 5.00(1H, br s), 4.37 (2H, d, J 5.8), 1.46 (9H, s); δ_(C) (101 MHz, CDCl₃)155.9, 144.7, 132.4, 127.8, 118.9, 111.1, 80.1, 44.2, 28.4; LRMS (ESI⁺):m/z 233 (100% [M+H]⁺).

Tetrazine 10 was synthesised by modification of a literature procedure.⁴Hydrazine monohydrate (1.024 mL, 21.10 mmol) was added to a stirringsuspension of tert-butylcarbamate S22 (98 mg, 0.44 mmol), formamidineacetate (439 mg, 4.22 mmol), and Zn(OTf)₂ (77 mg, 0.22 mmol) in1,4-dioxane (0.5 mL) at room temperature. The reaction was heated to 60°C. and stirred for 16 h. The reaction was cooled to room temperature anddiluted with EtOAc (10 mL). The reaction was washed with 1M HCl (10 mL)and the aqueous phase extracted with EtOAc (2×5 mL). The organic phasewas dried over sodium sulfate, filtered and evaporated under reducedpressure. The resulting crude residue was dissolved in a mixture of DCMand acetic acid (1:1, 5 mL), and NaNO₂ (584 mg, 8.44 mmol) was addedslowly over a period of 15 minutes, during which time the reactionturned bright red. The nitrous fumes were chased with an active airpurge and the reaction then diluted with DCM (25 mL). The reactionmixture was washed with sodium bicarbonate (sat., aq., 25 mL) and theaqueous phase extracted with DCM (2×10 mL). The organic phase was driedover sodium sulfate, filtered and evaporated under reduced pressure. Theresulting residue was purified by silica gel chromatography (20% EtOAcin hexane) to yield tetrazine 10 as a pink solid (85 mg, 70%). δ_(H)(400 MHz, CDCl₃) 10.21 (1H, s), 8.60 (2H, d, J 8.2), 7.53 (2H, d, J8.2), 4.97 (1H, br s), 4.45 (2H, d, J 6.0), 1.49 (9H, s); δ_(C) (101MHz, CDCl₃) 149.4, 142.6, 141.1, 132.1, 120.8, 119.2, 118.8, 51.8, 39.0;LRMS (ESI⁺): m/z 188 (100% [(M-Boc)+2H]⁺). 4M HCl in dioxane (2 mL, 8.0mmol) was added to a stirring solution of tetrazine 10 (75 mg, 0.26mmol) in DCM (4 mL). After 1 h the reaction was complete and the solventwas removed under reduced pressure to yield primary amine hydrochlorideS23 as a pink solid (61 mg, 100%). δ_(H) (400 MHz, d₆-DMSO) 10.64 (1H,s), 8.54 (2H, d, J 8.4), 7.79 (2H, d, J 8.4), 4.18 (2H, d, J 5.5); δ_(C)(101 MHz, d₆-DMSO) 165.2, 158.2, 138.9, 131.9, 129.8, 127.9, 41.8; LRMS(ESI⁺): m/z 188 (100% [M+H]⁺).

E-5-hydroxycyclooctene and E-exo-Bicyclo[6.1.0]non-4-ene-9-ylmethanolwere either made by previously described photochemical procedures^(5,6),or by the non-photochemical protocols described below.

Diisobutylaluminium hydride (1.0 M solution in cyclohexane, 89 mL, 89mmol) was added drop-wise to a stirring solution of commerciallyavailable 9-oxabicyclo[6.1.0]non-4-ene S24 (10 g, 80.53 mmol) in DCM(300 mL) at 0° C. The solution was stirred at 0° C. for 30 min, warmedto room temperature and stirred for 16 h. After this time, the reactionwas cooled to 0° C. and propan-2-ol (50 mL) was added slowly followed byHCl (1M, aq., 100 mL). The aqueous phase was extracted with DCM (3×200mL). The combined organics were washed with brine, dried over sodiumsulfate, filtered and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography (10-20% EtOAc inhexanes) to yield cyclooctene-4-ol S25 as a colorless oil (8.42 g, 83%).Spectral data was in accordance with the literature.⁷

tert-Butyl(chloro)dimethylsilane (13.3 g, 88.0 mmol) was added to astirring solution of cyclooctene-4-ol S25 (5.6 g, 44.0 mmol), imidazole(7.5 g, 0.11 mol) and DMAP (1 crystal) in DCM (30 mL) at 0° C. Thesolution was warmed to room temperature and stirred for 90 min, duringwhich time a white precipitate formed. The reaction was cooled to 0° C.,diluted with DCM (100 mL) and sodium bicarbonate (sat., aq., 100 mL) wasadded. The phases were separated and the aqueous phase was extractedwith DCM (3×100 mL). The combined organics were washed with brine (200mL), dried over sodium sulfate, filtered and concentrated under reducedpressure. The crude material was purified by silica gel chromatography(10-20% DCM in hexane) to yield silyl ether S26 as colorless oil (10.55g, quant.). δ_(H) (400 MHz, CDCl₃) 5.71-5.63 (1H, m), 5.60-5.52 (1H, m),3.80 (11, app td, J 8.6, 4.2), 2.34 (1H, dtd, J 13.8, 8.2, 3.8),2.25-2.15 (1H, m), 2.13-2.05 (1H, m), 2.02-1.93 (1H, m), 1.87-1.52 (5H,m), 1.47-1.35 (1H, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); δ_(C)(101 MHz, CDCl₃) 130.4, 129.4, 73.1, 38.0, 36.5, 26.1, 25.8, 25.1, 22.7,18.4, −3.4; LRMS (ESI⁺): m/z 241 (11% [M+H]⁺).

Peracetic acid (39% in acetic acid, 10.3 ml, 52.7 mmol) was addeddrop-wise to a stirred solution of silyl ether S26 (10.6 g, 43.9 mmol)and sodium carbonate (7.0 g, 65.8 mmol) in DCM (80 mL) at 0° C. Themixture was warmed to room temperature and stirred for 14 h. Thereaction was cooled to 0° C., diluted with DCM (50 mL) and sodiumthiosulfate (sat., aq., 100 mL) was added. The mixture was stirred atroom temperature for 10 min and then basified to pH 12 with NaOH (2M,aq.). The phases were separated and the organic phase washed with H₂O(100 mL), brine (100 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude material was purified bysilica gel chromatography (80%-90% DCM in hexane) to yield epoxidesS27/S28, as an inseparable mixture of diastereomers (2.3:1 by ¹H-NMR)and as a colorless oil (10.2 g, 91%). Major diastereomer: δ_(H) (400MHz, CDCl₃) 3.90 (1H, app sext, J 4.2), 2.90 (2H, ddd, J 16.7, 8.3,4.4), 2.21-2.09 (1H, m), 1.85-1.60 (6H, m), 1.50-1.38 (2H, m), 1.34-1.23(1H, m), 0.88 (9H, s), 0.04 (3H, s), 0.03 (3H, s); δ_(C) (101 MHz,CDCl₃) 171.9, 55.5, 55.4, 36.3, 34.3, 27.7, 26.0, 25.8, 22.6, 18.3,−3.4; LRMS (ESI⁺): m/z 257 (8% [M+H]⁺).

n-Butyllithium (2.5 M in hexanes, 14.8 mL, 37.0 mmol) was addeddrop-wise over 15 min to a stirring solution of epoxides S27/S28 (7.9 g,30.8 mmol) and diphenylphosphine (6.43 mL, 37.0 mmol) in THF (80 mL) at−78° C. The resulting mixture was stirred at −78° C. for 1 h, warmed toroom temperature and stirred for 14 h. The reaction mixture was dilutedwith THF (80 mL) and cooled to 0° C. Acetic acid (5.54 mL, 92.4 mmol)was added followed by hydrogen peroxide (30% solution in H₂O, 7.68 mL,67.7 mmol). The reaction mixture was warmed to room temperature andstirred for 4 h. Sodium thiosulfate (sat., aq., 100 mL) was added andthe mixture stirred for 10 min. The aqueous phase was extracted withEtOAc (3×200 mL). The combined organics were washed with brine (3×200mL), dried over sodium sulfate, filtered and concentrated under reducedpressure to yield phosphine oxides S29/S30/S31/S32 as a mixture of fourdiastereomers, which were used without further purification. δ_(P) (162MHz, CDCl₃) 45.2, 44.8, 44.4, 43.8; LRMS (ESI⁺): m/z 459 (100% [M+H]⁺).

Sodium hydride (60% dispersion in mineral oil, 2.46 g, 61.5 mmol) wasadded to a stirring solution of crude hydroxyl phosphine oxidesS29/S30/S31/S32 in DMF (100 mL) at 0° C. The resulting mixture waswarmed to room temperature, wrapped in tin foil and stirred for 2 h. Thereaction was cooled to 0° C., diluted with Et₂O (200 mL) and H₂O (200mL) was added. The phases were separated and the combined organicswashed with brine (2×200 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude mixture was purified bysilica gel chromatography (1-15% DCM in hexane) to yieldtrans-cyclooctenes S33/S34 as a separable mixture of diastereomers, withexclusive E-selectivity, and as colorless oils (2.78 g, 1.2:1 dr, 38%over 3 steps). S33: δ_(H) (400 MHz, CDCl₃) 5.64 (1H, ddd, J 16.0, 10.8,3.6), 5.45 (1H, ddd, J 15.9, 11.1, 3.2), 4.01 (1H, app dd, J 10.2, 5.4),2.41 (1H, qd, J 11.5, 4.4), 2.26-2.19 (1H, m), 2.09-1.94 (3H, m),1.92-1.73 (2H, m), 1.71-1.63 (1H, m), 1.54 (1H, tdd, J 14.0, 4.7, 1.1),1.30-1.08 (1H, m), 0.94 (9H, s), 0.03 (3H, s), 0.01 (3H, s); δ_(C) (101MHz, CDCl₃) 135.9, 131.5, 67.6, 44.0, 35.2, 34.8, 29.7, 27.7, 26.2,18.4, −4.7, −4.8; LRMS (ESI⁺): m/z 241 (8% [M+H]⁺). S34: δ_(H) (400 MHz,CDCl₃) 5.55 (1H, ddd, J 15.9, 11.0, 3.6), 5.36 (1H, ddd, J 16.1, 10.8,3.4), 3.42-3.37 (1H, m), 2.36-2.28 (2H, m), 2.22 (1H, app qd, J 11.2,6.3), 2.02-1.87 (4H, m), 1.73 (1H, dd, J 14.9, 6.2), 1.67-1.45 (2H, m),0.87 (9H, s), 0.03 (6H, s); δ_(C) (101 MHz, CDCl₃) 135.5, 132.5, 78.6,44.9, 42.0, 34.6, 33.0, 31.3, 26.1, 18.3, −4.4, −4.5; LRMS (ESI⁺): m/z241 (12% [M+H]⁺). For all further experiments trans-cyclooctene S34 wasused, where the C4-oxygen substituent occupies an equatorial position.

Tetrabutylammonium fluoride (1M solution in THF, 23.8 mL. 23.8 mmol) andcesium fluoride (1.08 g, 7.14 mmol) were added to a stirring solution ofsilyl ether S34 (573 mg, 2.38 mmol) in MeCN (5 mL) at room temperature.The resulting mixture was wrapped in tin foil and stirred at roomtemperature for 36 h. After this period the reaction was cooled to 0°C., diluted with DCM (100 mL) and H₂O (100 mL) was added. The phaseswere separated, the organic phase washed with brine (2×100 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure.The crude material was purified by silica gel chromatography (20% EtOAcin hexane) to yield secondary alcohol S35 as a colorless oil (289 mg,96%) δ_(H) (400 MHz, CDCl₃) 5.60 (1H, ddd, J 16.0, 10.7, 4.2), 5.41 (1H,ddd, J 16.0, 11.1, 3.7), 3.52-3.45 (2H, m), 2.40-2.25 (3H, m), 2.03-1.90(4H, m), 1.75-1.53 (3H, m), 1.25-1.18 (1H, m); δ_(C) (101 MHz, CDCl₃)135.1, 132.8, 77.7, 44.6, 41.1, 34.3, 32.6, 32.1; LRMS (ESI⁺): m/z 127(14% [M+H]⁺).

Succimidyl carbonate S36 (200 mg, 0.75 mmol) was added to a stirringsolution of Fmoc-Lys-OH.HCl (303 mg, 0.75 mmol) and DIPEA (0.19 g, 1.50mmol) in DMF (7.5 mL) at 0° C. The solution was warmed to roomtemperature, wrapped in tin foil and stirred for 12 h. After this periodthe solution was concentrated under reduced pressure and purified bysilica gel chromatography (0-10% MeOH in DCM) to yield Fmoc-TCOK-OHS37/S38 as a yellow oil that still contained DMF (350 mg, 81%). δ_(H)(400 MHz, CDCl₃) 7.75-7.69 (2H, m), 7.63-7.52 (2H, m), 7.41-7.33 (2H,m), 7.32-7.25 (2H, m), 5.82-5.34 (3H, m), 5.27 (1H, br s), 4.90-4.50(1H, m), 4.47-4.01 (5H, m), 3.32-3.30 (1H, m), 2.39-1.08 (17H, m); δ_(C)(100 MHz, CDCl₃) 174.3, 156.3, 155.9, 143.8, 143.6, 141.1, 135.0, 134.8,132.8, 132.6, 127.5, 126.9, 125.0, 119.8, 80.3, 66.8, 53.4, 47.0, 41.0,40.4, 38.5, 34.1, 32.5, 32.3, 32.1, 30.8, 29.3, 22.3; ESI-MS (m/z):[M+Na]+ calcd. for C₃₀H₃₆N₂O₆Na 543.2471. found 543.2466.

Piperidine (1 mL) was added to a stirring solution of Fmoc-TCOK-OHS37/S38 (0.269 g, 0.517 mmol) in DCM (4 mL). The mixture was wrapped intin foil and stirred at room temperature for 30 min. The reactionmixture was concentrated under reduced pressure and the crude materialwas purified by silica gel chromatography (30-50% MeOH in DCM) to yieldH-TCOK-OH 1 as an ivory-colored solid. δ_(H) (400 MHz, d₄-MeOD)5.63-5.56 (1H, m), 5.50-5.43 (1H, m), 4.31-4.25 (1H, m), 3.60-3.53 (1H,m), 3.11-3.03 (2H, m), 2.37-2.26 (3H, m), 2.02-1.36 (13H, m); δ_(C) (100MHz, d₄-MeOD) 174.3, 159.0, 136.3, 133.9, 81.8, 56.0, 42.4, 41.4, 39.8,35.4, 33.7, 32.3, 32.1, 30.9, 23.6; ESI-MS (m/z): [M−H]⁻ calcd. forC₁₅H₂₅N₂O₄ 297.1814. found 297.1811.

exo-Bicyclo[6.1.0]non-4-ene-9-ylmethanol S18 was synthesised accordingto a literature procedure.⁵

tert-Butyl(chloro)diphenylsilane (7.45 g, 27.1 mmol) was added to astirring solution of exo-bicyclo[6.1.0]non-4-ene-9-ylmethanol S18 (2.75g, 18.1 mmol), imidazole (2.15 g, 31.6 mmol) and DMAP (2.21 g, 18.1mmol) in DCM (35 ml) at 0° C. The solution was warmed to roomtemperature and stirred for 24 h, during which a white precipitateformed. The reaction was cooled to 0° C., diluted with DCM (100 mL) andsodium bicarbonate (sat., aq., 100 mL) was added. The phases wereseparated and the aqueous phase was extracted with DCM (3×100 mL). Thecombined organics were washed with brine (200 mL), dried over sodiumsulfate, filtered and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography (20% DCM in hexane)to yield silyl ether S39 as a colorless oil (6.85 g, 97%), δ_(H) (400MHz, CDCl₃) 7.79-7.64 (4H, m), 7.50-7.32 (6H, m), 5.63 (2H, dm, J 11.5),3.59 (2H, d, J 6.2), 2.40-2.21 (2H, m), 2.18-1.96 (4H, m), 1.45-1.33(2H, m), 1.07 (9H, s), 0.72-0.56 (3H, m); δ_(C) (101 MHz, CDCl₃) 135.7,134.3, 130.2, 129.5, 127.6, 67.9, 29.1, 28.6, 27.2, 26.9, 22.0, 19.3;LRMS (ESI⁺): m/z 408 (10%, [M+NH₄]⁺).

Peracetic acid (3.38 ml, 39% in acetic acid, 19.9 mmol) was added to astirred solution of silyl ether S39 (6.49 g, 16.6 mmol) and anhydroussodium carbonate (2.64 g, 24.9 mmol) in DCM (65 mL) at 0° C. The mixturewas warmed to room temperature and stirred for 24 h. The reaction wasthen cooled to 0° C., diluted with DCM (100 mL) and sodium thiosulfate(sat., aq., 150 mL) was added. The mixture was stirred at roomtemperature for 30 min and then basified to pH 12 with NaOH (2M, aq.,).The phases were separated and the organic phase was washed with H₂O (200mL), brine (200 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude material was purified bysilica gel chromatography (100% DCM) to yield epoxides S40 and S41 as aninseparable mixture of diastereomers (1:1 by ¹H NMR spectroscopy) and asa colorless oil (5.97 g, 88%). δ_(H) (400 MHz, CDCl₃) 7.72-7.63 (8H, m),7.47-7.34 (12H, m), 3.57 (2H, d, J 5.6), 3.54 (2H, d, J 5.9), 3.03-3.10(2H, m), 3.02-2.91 (2H, m), 2.36-2.24 (2H, m), 2.21-2.08 (2H, m),2.06-1.85 (6H, m), 1.35-1.12 (4H, m), 1.06 (9H, s), 1.05 (9H, s),0.92-0.80 (2H, m), 0.78-0.47 (6H, m); δ_(C) (101 MHz, CDCl₃) 135.65,135.63, 134.2, 134.1, 129.6 (2×CH), 127.6 (2×CH), 67.4, 67.0, 56.91,56.85, 29.7, 27.7, 26.9 (2×3CH₃), 26.6, 26.5, 23.31, 23.25, 21.7, 20.4,19.2 (2×2C); LRMS (ESI⁺): m/z 407 (9%, [M+H]⁺).

n-Butyllithium (2.5 M in hexanes, 5.92 mL, 14.8 mmol) was added dropwise over 15 min to a stirring solution of epoxides S40/S41 (5.47 g,13.5 mmol) and diphenylphosphine (2.57 mL, 14.80 mmol) in THF (50 mL) at−78° C. The resulting mixture was stirred at −78° C. for 1 h, warmed toroom temperature and stirred for additional 14 h. The reaction mixturewas diluted with THF (80 mL) and cooled to 0° C. Acetic acid (1.54 mL,26.9 mmol) was added followed by addition of hydrogen peroxide (30%solution in H₂O, 3.05 mL, 26.9 mmol). The reaction mixture was warmed toroom temperature and stirred for 4 h. Sodium thiosulfate (sat., aq., 100mL) was added and the mixture stirred for 10 min. The aqueous phase wasextracted with EtOAc (3×200 mL). The combined organics were washed withbrine (3×200 mL), dried over sodium sulfate, filtered and concentratedunder reduced pressure. The crude mixture was purified by silica gelchromatography (40-100% EtOAc in hexane) to yield phosphine oxidesS42/S43/S44/S45 as a 51:18 mixture of two diasteroisomers (5.61 g, 69%over 2 steps), each of which is a 1:1 mixture of regioisomers (S42/S45and S43/S44). Major diastereomer: δ_(H) (400 MHz, CDCl₃) 7.82-7.68 (4H,m), 7.68-7.58 (4H, m), 7.52-7.32 (12H, m), 4.58-4.45 (1H, m), 4.16 (1H,d, J 5.3), 3.54 (2H, d, J 6.0), 2.47 (1H, ddd, J 12.0, 11.7, 4.3),2.21-2.07 (1H, m), 2.05-1.85 (2H, m), 1.78-1.55 (3H, m), 1.22-1.05 (1H,m), 1.03 (9H, s), 0.91-0.75 (1H, m), 0.62-0.35 (3H, m); δ_(P) (162 MHz,CDCl₃) 39.7; LRMS (ESI⁺): m/z 609 [100%, (M+H)⁺]. Minor diastereomer:δ_(H) (400 MHz, CDCl₃) 7.87-7.77 (2H, m), 7.74-7.60 (6H, m), 7.52-7.30(12H, m), 4.26 (1H, d, J 4.0), 3.89-3.78 (1H, m), 3.63 (1H, dd, J 10.7,5.8), 3.54 (1H, dd, J 10.7, 6.2), 3.26-3.10 (1H, m), 2.22-2.12 (1H, m),2.00-1.78 (3H, m), 1.70-1.62 (1H, m), 1.42-1.28 (1H, m), 1.04 (9H, s),1.04-0.92 (2H, m), 0.79-0.65 (1H, m), 0.55-0.41 (1H, m), 0.27-0.12 (1H,m); δ_(P) (162 MHz, CDCl₃) 39.6; LRMS (ESI⁺): m/z 609 [100%, (M+H)⁺].

Sodium hydride (60% dispersion in mineral oil, 0.46 g, 11.5 mmol) wasadded to a stirring solution of hydroxyl phosphine oxidesS42/S43/S44/S45 (4.68 g, 7.69 mol) in anhydrous DMF (60 mL) at 0° C. Theresulting mixture was warmed to room temperature, wrapped in tin foiland stirred for 2 h. The reaction mixture was cooled to 0° C., dilutedwith Et₂O (200 mL) and H₂O (200 mL), the phases were separated andaqueous phase was extracted with hexane (150 mL). The combined organicswere washed with brine (sat., aq., 5×250 mL), dried over sodium sulfate,filtered and concentrated under reduced pressure. The crude mixture waspurified by silica gel chromatography (1-20% DCM in hexane) to yieldtrans-cyclooctene S46 as a single diastereomer and with exclusiveE-selectivity (2.08 g, 69%); δ_(H) (400 MHz, CDCl₃) 7.72-7.62 (4H, m),7.46-7.34 (6H, m), 5.83 (1H, ddd, J 16.1, 9.2, 6.2), 5.11 (1H, ddd, J16.1, 10.6, 3.3), 3.59 (2H, d, J 5.7), 2.28-2.40 (1H, m), 2.12-2.27 (3H,m), 1.80-1.95 (2H, m), 1.04 (9H, s), 0.74-0.90 (1H, m), 0.46-0.60 (1H,dm, J 14.0), 0.31-0.42 (2H, m), 0.18-0.29 (1H, m); δ_(C) (101 MHz,CDCl₃) 138.6, 135.8, 134.4, 131.3, 129.6, 127.7, 68.1, 39.0, 34.1, 32.9,28.2, 27.9, 27.0, 21.6, 20.5, 19.4,

Tetrabutylammonium fluoride (1M solution in THF, 10.0 ml, 10.0 mmol) wasadded to a stirring solution of silyl ether S46 (0.78 g, 2 mmol) in THF(5 mL) at room temperature, wrapped in tin foil and stirred for 45 min.After this period, the reaction mixture was concentrated under reducedpressure, diluted with DCM (100 mL) and washed with brine (100 mL). Thephases were separated and the organic phase washed with brine (2×100mL). The combined organics were dried over sodium sulfate, filtered andconcentrated under reduced pressure. The crude material was purified bysilica gel chromatography (20% EtOAc in hexane) to yield primary alcoholS47 as a colorless oil (0.29 g, 96%); δ_(H) (400 MHz, d₄-MeOD) 5.87 (1H,ddd, J 16.5, 9.3, 6.2), 5.13 (1H, dddd, J 16.5, 10.4, 3.9, 0.8),3.39-3.47 (2H, dd, J 6.2, 1.5), 2.34-2.44 (1H, m), 2.12-2.33 (3H, m),1.82-1.98 (2H, m), 0.90 (1H, dtd, J 12.5, 12.5, 7.1), 0.55-0.70 (1H, m),0.41-0.55 (1H, m), 0.27-0.41 (2H, m); δ_(C) (101 MHz, d₄-MeOD) 139.3,132.2, 67.5, 39.9, 34.8, 33.8, 29.2, 28.7, 23.0, 21.9; MS-CI (NH₃): m/z[M-OH] calcd. for C₁₀H₁₅, 135.1174. found 135.1173.

pNO₂-phenyl carbonate S48 (250 mg, 0.79 mmol) was added to a stirringsolution of Fmoc-Lys-OH.HCl (478 mg, 1.18 mmol) and DIPEA (0.27 mL, 1.58mmol) in DMF (3 mL) at 0° C. The solution was warmed to roomtemperature, wrapped in tin foil and stirred for 16 h. After this periodthe solution was concentrated under reduced pressure and purified bysilica gel chromatography (0-5% MeOH in DCM) to yield Fmoc-exo-sTCOK S49as a white foam (373 mg, 87%). δ_(H) (400 MHz, d₆-DMSO) 13.09-12.06 (1H,br s), 7.90 (2H, d, J 7.5), 7.73 (2H, d, J 7.5), 7.66-7.56 (1H, m), 7.43(2H, t, J 7.4), 7.34 (2H, J 7.4), 7.08 (1H, t, J 5.4), 5.84-5.72 (1H,m), 5.13-5.01 (1H, m), 4.31-4.19 (3H, m), 3.93-3.79 (3H, m), 3.00-2.90(2H, m), 2.31-2.07 (4H, m), 1.91-1.78 (2H, m), 1.75-1.49 (2H, m),1.45-1.22 (4H, m), 0.91-0.75 (1H, m), 0.62-0.45 (2H, m), 0.43-0.32 (2H,m); δ_(C) (101 MHz, d₆-DMSO) 173.9, 156.4, 156.1, 143.8, 140.7, 137.9,131.0, 127.6, 127.0, 125.2, 120.1, 79.1, 67.9, 65.6, 53.8, 46.6, 38.1,33.4, 31.9, 30.4, 29.0, 27.2, 24.3, 22.8, 21.2, 20.2; LRMS (ESI⁺): m/z545 (100% [M−H]⁻).

Lithium hydroxide monohydrate (94 mg, 0.75 mmol) was added to a stirringsolution of exo-sTCOK S49 in THF:H₂O (3:1, 8 mL). The solution waswrapped in tin foil, stirred for 4 h at room temperature and EtOAc (100mL) and H₂O (100 mL) were added. The aqueous phase was carefullyacidified to pH 4 by the addition of AcOH and extracted with EtOAc(4×100 mL). The aqueous phase was evaporated under reduced pressure andfreeze-dried to yield exo-sTCOK 3 as a white solid. For all subsequentlabeling experiments using mammalian cells exo-H-bcnK-OH 1 was furtherpurified by reverse-phase HPLC (0:1 H₂O:MeCN to 9:1 H₂O:MeCN gradient).δ_(H) (400 MHz, d₆-DMSO) 7.21-7.09 (1H, br m), 5.85-5.72 (1H, m),5.14-5.02 (1H, m), 3.80 (2H, d, J 2.6), 3.14-3.05 (1H, m), 2.98-2.86(2H, m), 2.31-2.08 (4H, m), 1.92-1.78 (2H, m), 1.73-1.65 (1H, m),1.55-1.44 (1H, m), 1.41-1.25 (4H, m), 0.90-0.62 (1H, m), 0.65-0.45 (2H,m), 0.43-0.32 (2H, m); δ_(C) (101 MHz, d₆-DMSO) 175.5, 156.3, 137.9,131.1, 67.8, 54.5, 38.1, 33.4, 32.1, 32.0, 29.2, 27.2, 24.7, 24.3, 22.5,21.2, 20.2; LRMS (ESI⁺): m/z 325 (100% [M+H]⁺).

REFERENCES TO SUPPLEMENTARY EXAMPLES

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REFERENCES TO MAIN TEXT

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All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed aspects and embodiments of the present invention will beapparent to those skilled in the art without departing from the scope ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are apparent tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A polypeptide comprising an amino acid having abicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group.
 2. A polypeptideaccording to claim 1 wherein said BCN group is present as a residue of alysine amino acid.
 3. A method of producing a polypeptide comprising aBCN group, said method comprising genetically incorporating an aminoacid comprising a BCN group into a polypeptide.
 4. A method according toclaim 3 wherein producing the polypeptide comprises (i) providing anucleic acid encoding the polypeptide which nucleic acid comprises anorthogonal codon encoding the amino acid having a BCN group; (ii)translating said nucleic acid in the presence of an orthogonal tRNAsynthetase/tRNA pair capable of recognising said orthogonal codon andincorporating said amino acid having a BCN group into the polypeptidechain.
 5. A method according to claim 3 wherein said amino acidcomprising a BCN group is a BCN lysine.
 6. A method according to claim 4wherein said orthogonal codon comprises an amber codon (TAG), said tRNAcomprises MbtRNA_(CUA), said amino acid having a BCN group comprises abicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine and said tRNAsynthetase comprises a PylRS synthetase having the mutations Y271M,L274G and C313A (BCNRS).
 7. A polypeptide according to claim 1, or amethod according to any of claims 3 to 6, wherein said amino acid havinga BCN group is incorporated at a position corresponding to a lysineresidue in the wild type polypeptide.
 8. A polypeptide according toclaim 1 which comprises a single BCN group.
 9. A polypeptide accordingto claim 1 wherein said BCN group is joined to a tetrazine group.
 10. Apolypeptide according to claim 9 wherein said tetrazine group is furtherjoined to a fluorophore.
 11. A polypeptide according to claim 10 whereinsaid fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) orboron-dipyrromethene (BODIPY).
 12. An amino acid comprisingbicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN).
 13. An amino acid accordingto claim 12 which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine.14. BCN lysine according to claim 13 having the structure:


15. A method of producing a polypeptide comprising a tetrazine group,said method comprising providing a polypeptide according to claim 1,contacting said polypeptide with a tetrazine compound, and incubating toallow joining of the tetrazine to the BCN group by an inverse electrondemand Diels-Alder cycloaddition reaction.
 16. A method according toclaim 15 wherein the tetrazine is selected from 6 to 17 of FIG.
 1. 17. Amethod according to claim 15 wherein the tetrazine is selected from 6,7, 8 and 9 of FIG. 1 and the pseudo first order rate constant for thereaction is at least 80 M⁻¹ s⁻¹.
 18. A method according to claim 15wherein said reaction is allowed to proceed for 10 minutes or less. 19.A method according to claim 18 wherein said reaction is allowed toproceed for 1 minute or less.
 20. A method according to claim 19 whereinsaid reaction is allowed to proceed for 30 seconds or less.
 21. A methodaccording to claim 15 wherein said tetrazine compound is a tetrazinecompound selected from the group consisting of 11 and 17 of FIG.
 1. 22.(canceled)
 23. (canceled)
 24. (canceled)